Retention of critical platelet hemostatic functions of amotosalen‐UVA pathogen‐reduced cryoprecipitated fibrinogen complex

Abstract

Background

Cryoprecipitate Anti‐Haemophilic Factor (CRYO‐AHF) is enriched for fibrinogen, VWF, FVIII, FXIII and fibronectin, but has short post‐thaw expiration due to risk of transfusion‐transmitted infection (TTI) limiting availability for rapid treatment. Amotosalen‐UVA pathogen reduction treatment (A‐PRT) to manufacture pathogen‐reduced cryoprecipitated fibrinogen complex (PRCFC) allows 5‐day post‐thaw expiration, reduces TTI risk, and facilitates early treatment of hemorrhage.

Aims

To evaluate adhesive protein functions in PRCFC and lyophilized PRCFC (LIFC).

Methods

CRYO‐AHF, PRCFC, LIFC, and commercial fibrinogen concentrate (CFC) were evaluated for platelet adhesion and aggregation in variable shear microfluidic assays.

Results

Platelet adhesion kinetics to CRYO‐AHF, PRCFC, and LIFC, under low shear flow (300 s⁻¹) were conserved. Platelet adhesion to CFC at low shear was reduced due to absence of functional VWF. αIIbβ3 integrin/fibrinogen and GPIb‐IX‐V/VWF platelet interactions with CRYO‐AHF, PRCFC, and LIFC were confirmed by abciximab and caplacizumab inhibition, respectively. All fibrinogen sources promoted efficient platelet aggregation. Perfusion of reconstituted plasma‐free blood (RBC + platelets + various cryoprecipitates) on immobilized VWF‐binding peptide (1500 s⁻¹) showed impaired platelet adhesion to CFC compared to PRCFC and CRYO‐AHF. Perfusion of reconstituted blood on collagen (3000 s⁻¹) indicated CRYO‐AHF, PRCFC and LIFC formed thrombi to similar levels. Platelets treated with A‐PRT combined with PRCFC or LIFC retained similar activity to CRYO‐AHF for platelet aggregation and thrombus formation on collagen.

Conclusions

PRCFC and LIFC retained critical hemostatic functions of VWF and fibrinogen to support platelet adhesion and aggregation during physiologic shear. PRCFC and LIFC represent a therapeutic option for early treatment of massive hemorrhage.

1. INTRODUCTION

Cryoprecipitate Anti‐Haemophilic Factor (Cryo‐AHF), obtained by specific plasma freezing–thawing methods, ¹ is enriched in fibrinogen, von Willebrand factor (VWF), Factor XIII, and Fibronectin. Cryo‐AHF is indicated to correct hypofibrinogenemia associated with massive bleeding during trauma, obstetric hemorrhage, major surgery such as cardiac surgery or liver transplantation. ¹ In addition to this primary indication, it is indicated to treat congenital fibrinogen deficiency, congenital FXIII deficiency, or von Willebrand disease if other products are not available. ² , ³ A major impediment to the use of Cryo‐AHF is the post‐thaw expiration time limited to 4–6 h, due to the potential risk of bacterial proliferation and transfusion‐transmitted infection (TTI). ⁴ , ⁵ Cryo‐AHF is used in the US and Canada ⁶ , ⁷ but is less widespread in Europe, mainly due to the lack of a pathogen‐inactivation process, and because the short post‐thaw expiration limits availability for transfusion. The availability of pathogen‐inactivation treatment during the manufacture of commercial purified fibrinogen concentrates (CFC) facilitated the movement away from Cryo‐AHF to generate fibrinogen concentrates with reduced TTI risk. However, fibrinogen concentrates and Cryo‐AHF vary in composition.

In order to reduce the risk of TTI, measures have been implemented, such as pathogen‐reduction treatment (PRT). ⁸ Today, PRT is widely used for manufacture of fresh frozen plasma and platelet concentrates to reduce TTI risk. Some PRT methods degrade pathogen membranes, such as the solvent/detergent method, while others inhibit pathogen nucleic acid replication. Blue methylene with the THERAFLEX MB PLASMA® technology (Macopharma, Mouvaux, France), or Amotosalen‐UVA with the INTERCEPT™ technology (Cerus Corporation, Concord, CA, USA) is in use. ⁹ , ¹⁰ , ¹¹ , ¹² Amotosalen is a psoralen, which intercalates between helical strands of DNA/RNA. UVA illumination (320–400 nm) induces adduct formation and cross‐linking of nucleic acids inhibiting pathogen replication. This process is efficient for inactivation of a spectrum of pathogens containing nucleic acid, and residual leukocytes, causing transfusion adverse effects. After UVA illumination, residual Amotosalen is substantially removed by a Compound Adsorption Device. ¹³

Cryo‐AHF is not manufactured with pathogen inactivation. However, to improve the safety of cryoprecipitates manufactured in blood centers and extend duration of utilization, the FDA approved Amotosalen‐UVA pathogen‐reduction treatment (A‐PRT) for the manufacture of Pathogen‐Reduced Cryoprecipitated Fibrinogen Complex (PRCFC). An advantage of PRCFC is that it can be used up to 5 days after thawing with room temperature storage due to the pathogen inactivation. ⁶ However, preparation of A‐PRT for PRCFC production results in a slight reduction of fibrinogen concentration, FVIII concentration, and VWF concentration with potential impact on hemostatic efficacy. ⁶ , ¹⁴ Despite the impact on coagulation factor content, its ability to promote the plasma phase of coagulation has been reported to be similar to Cryo‐AHF. ¹⁵ , ¹⁶ In addition to the plasma phase of coagulation, the interactions of platelets with VWF and fibrinogen are important to form a platelet thrombus and stop bleeding. ¹⁷ Vessel rupture leads to hemorrhage, during which VWF binds to the sub‐endothelium, unfolds and allows initial adhesion of platelets through the GPIb‐IX‐V complex. ¹⁸ This is a central step, as shown by patients with functional VWF defects, who have increased bleeding risk. ¹⁹ In addition, following adhesion and activation, platelets aggregate by interacting with fibrinogen via integrin αIIbβ3. ²⁰ This interaction is central for clot formation and hemostasis, as demonstrated by hypofibrinogenemic patients who have increased hemorrhagic risk. ²¹ Thus, the functional maintenance of these proteins during cryoprecipitate manufacture is essential for effective treatment of hemorrhage.

The objective of our study is to evaluate the effect of A‐PRT on the ability of PRCFC to retain VWF and fibrinogen‐mediated platelet adhesion and aggregation properties under flow conditions. Evidence that PRCFC retains VWF and fibrinogen hemostatic function supports use as a therapeutic option for early treatment of massive hemorrhage.

2. METHODS

2.1. In vitro microfluidic flow assays

Microfluidic flow chambers were prepared as described. ²² Channels were coated with cryoprecipitates (Cryo‐AHF, PRCFC, LIFC), and Commercial Fibrinogen Complex (CFC, Octapharma) adjusted to 3 mg/mL of fibrinogen in PBS and incubated for 2 h at room temperature. Additional microfluidic flow chambers were coated with a peptide of collagen containing the vWF binding site (VWF‐binding peptide, CambCol Laboratories) adjusted to 100 μg/mL and incubated overnight at 4°C. Other microfluidic flow chambers were incubated with type I fibrillar collagen at 200 μg/mL (Takeda) during 1 h at room temperature. Microfluidic channels were passivated with human serum albumin solution (10 mg/mL) in phosphate buffer saline for 30 min at room temperature as recommended by the ISTH biorheology subcommittee. ²³ Hirudinated (525 ATU/mL) whole blood was perfused over immobilized cryoprecipitates at 300 s⁻¹ during 5 min. Plasma‐free blood for chamber perfusion studies was composed of washed platelets or pathogen‐reduced washed platelets stored during 1 up to 7 days (1.0 × 10⁵/μL), washed RBCs (hematocrit of 40%), various cryoprecipitates (adjusted to 3 mg of fibrinogen/mL in Tyrode's albumin buffer) and hirudin (100 U/mL). Plasma‐free reconstituted blood was perfused at 1500 s⁻¹ over VWF‐binding peptide during 3 min and at 1500 or 3000 s⁻¹ through fibrillar type‐1 HORM collagen (equine Achilles tendon, Takeda) coated chambers during 10 and 5 min respectively. Different cap times were used for the flow assays with the objective to obtain relevant levels of platelet adhesion and thrombus formation reaching plateau values, all depending on the wall shear rate conditions used. The time difference is attributable to the use of stored pathogen‐reduced washed platelets, which exhibit reduced reactivity, particularly after 7 days of storage, compared to fresh platelets. This necessitated a reduction in shear rate and an extension of perfusion time to allow the formation of thrombi of sufficient size for analysis. For inhibition studies, the blood was incubated with abciximab (40 μg/mL), an αIIbβ3 antagonist, for 10 min at 37°C before perfusion, or with caplacizumab (10 μg/mL), a VWF antagonist added to the blood just prior to perfusion. Because abciximab and caplacizumab are used clinically, we selected 0.9% NaCl as a control, rather than isotype antibody control. Previous studies have shown that the use of a matched isotype control or buffer resulted in comparable platelet functional responses. ²⁴ , ²⁵ A programmable syringe pump (PHD 2000, Harvard Apparatus) was used to perfuse blood through the microfluidic chambers. To monitor platelet adhesion in real time, an inverted Leica microscope using a 40× 1.25 numerical aperture oil objective and a Hamamatsu CMOS ORCA FLASH‐4 LT camera (Hamamatsu Photonics, Massy, France) with differential interference contrast was used. At the end of the perfusion time, images were taken and the number of platelets/mm² was quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). To monitor thrombus formation on collagen, platelets were labeled with 1 μmol/L of DIOC₆ (Molecular Probes) for 10 min at 37°C, and fluorescence emission was measured with a 488 nm argon‐ion laser using a confocal Leica inverted microscope with a resonant scanner and a 40x oil objective. Optical sections in xyz from the base to the peak of the thrombi were recorded and a maximum intensity projection of the stacked images was performed with ImageJ software. The thrombi area (in μm²), corresponding to the fluorescent signal area, was analyzed.

2.2. Platelet aggregation assay

Platelet aggregation with plasma‐free washed platelets was measured during 5 min at 37°C using a turbidimetric light transmission method in an APACT 4004 aggregometer (LABiTEC, Ahrensburg, Germany). Cryoprecipitates (Cryo‐AHF, PRCFC, and LIFC) or fibrinogen concentrate adjusted to 30 μg of fibrinogen/mL were added to washed platelets adjusted to 3.0 × 10⁵/μL with activation by 5 μmol/L of ADP (MAST DIAGNOSTIC). The maximum level of platelet aggregation was quantified.

2.3. Statistical analysis

Statistical analysis was performed using GraphPad software (Prism 9.2.0). Data were reported as mean ± standard error of the mean. Parametric distribution of the data was established using the Shapiro–Wilk test. Depending on parametric distribution, data with parametric distributions were analyzed using one‐way ANOVA followed by Bonferroni post‐hoc test (Figures 1C,F, 2C, 3B, 4C, and 5C,E) or using Kruskall‐Wallis test for data with nonparametric distributions (Figure 5G). A p value of <0.05 was considered as statistically significant. Sample sizes are presented in figure legends.

FIGURE 1.

Evaluation of fibrinogen and von Willebrand Factor from cryoprecipitates and CFC to absorb to chamber surfaces and support platelet adhesion under flow. (A–C) Hirudinated whole blood with added NaCl 0.9% (Control) or abciximab (40 μg/mL) was perfused into microfluidic chambers coated with Cryo‐AHF (blue), PRCFC (red), LIFC (green), and CFC (purple), or 1% human serum albumin (negative control, black) at 300 s⁻¹ for 5 min (n = 4 blood donors, 1 preparation of Cryo‐AHF, PRCFC, LIFC, and CFC was used). (A) Representative images of the microfluidic chambers after 5 min of whole blood perfusion. Scale bar = 30 μm. (B) Kinetics of platelets adhesion with and without addition of abciximab. (C) Number of platelets adhering to coated microfluidic chambers per mm² (One‐way analysis of variance with Bonferroni post hoc test). (D–F) Hirudinated whole blood with added NaCl 0.9% (Control) or caplacizumab (10 μg/mL) was perfused into microfluidic chambers coated with Cryo‐AHF (blue), PRCFC (red), LIFC (green), and CFC (purple), or 1% human serum albumin (negative control, black) at 300 s⁻¹ for 5 min (n = 4 blood donors, 1 preparation of Cryo‐AHF, PRCFC, LIFC, and CFC was used). (D) Representative images of the microfluidic chambers after 5 min of whole blood perfusion. Scale bar = 30 μm. (E) Kinetics of platelets adhesion. (F) Number of platelets adhering to coated microfluidic chambers per mm² (one‐way analysis of variance with Bonferroni post hoc test).

FIGURE 2.

Evaluation of VWF from cryoprecipitates and CFC to absorb to a surface coated with VWF binding peptide and to support platelet adhesion under flow. (A–C) Reconstitued hirudinated blood (red blood cells + platelets + cryoprecipitates, CFC, or buffer) containing NaCl 0.9% (Control) or caplacizumab (10 μg/mL) was perfused into microfluidic chambers coated with VWF‐binding peptide at 1500 s⁻¹ for 3 min (n = 4 blood donors, 1 preparation of LIFC and CFC, 2 preparations of Cryo‐AHF and PRCFC were used). (A) Representative images of the microfluidic chambers after 3 min of blood perfusion. Scale bar = 30 μm. (B) Kinetics of platelet adhesion. (C) Number of platelets adhering to coated microfluidic chamber per mm² (one‐way analysis of variance, Bonferroni post hoc test).

FIGURE 3.

Evaluation of the functional quality of fibrinogen in cryoprecipitates and CFC to promote platelet aggregation. (A, B) Aggregation assays were performed with washed platelets from healthy blood donors containing fibrinogen (30 μg/mL) from cryoprecipitates or CFC (n = 3 blood donors, 1 preparation of Cryo‐AHF, PRCFC, LIFC, and CFC were used). (A) Representative aggregation curves induced by ADP (5 μM). (B) Maximum of aggregation induced by ADP (5 μM) (one‐way analysis of variance, Bonferroni post hoc test).

FIGURE 4.

Evaluation of cryoprecipitates and CFC to support thrombus formation on collagen coated surfaces under flow. (A–C) Reconstituted hirudinated plasma‐free blood (red blood cells + platelets + cryoprecipitates, CFC or buffer) was perfused into microfluidic chamber coated with collagen HORM (200 μg/mL) at 3000 s⁻¹ for 5 min (n = 5 blood donors, 1 preparation of Cryo‐AHF, PRCFC, LIFC, and CFC was used). (A) Representative confocal Z stack images of thrombi volumes on collagen coated surfaces after 5 min of perfusion at 3000 s⁻¹. Scale bar = 50 μm. (B) Kinetics of thrombi formation. (C) Surface area of thrombi at 5 min on collagen coated surfaces (One‐way analysis of variance, Bonferroni post hoc test).

FIGURE 5.

Evaluation of cryoprecipitates and CFC to support thrombus formation to collagen‐coated surfaces under flow with fresh and stored pathogen‐reduced buffy‐coat platelet components. (A–G) Reconstituted hirudinated plasma‐free blood (red blood cells + pathogen‐reduced platelets + cryoprecipitates, CFC, or buffer) was perfused into a microfluidic chamber coated with collagen HORM (200 μg/mL) at 1500 s⁻¹ for 10 min (n = 4 blood donors, 1 preparation of Cryo‐AHF, PRCFC, LIFC, and CFC was used). (A) Representative confocal Z stack images of thrombi volumes on collagen surfaces after 10 min of perfusion at 1500 s⁻¹ with platelets stored for 2, 5, or 7 days after A‐PRT. Scale bar = 50 μm. (B) Kinetics of thrombi formation with platelets perfused at 2 days of storage after A‐PRT. (C) Surface of thrombi at 10 min on collagen surfaces with platelets perfused 2 days after A‐PRT (one‐way analysis of variance, Bonferroni post hoc test). (D) Kinetics of thrombi formation with platelets perfused 5 days after pathogen reduction. (E) Surface of thrombi at 10 min on collagen surfaces with platelets perfused 5 days after pathogen reduction. (One‐way analysis of variance, Bonferroni post hoc test). (F) Kinetics of thrombi formation with platelets perfused 7 days after A‐PRT. (G) Surface of thrombi at 10 min on collagen surfaces with platelets perfused 7 days after pathogen reduction (Kruskall Wallis).

Detailed methods for production of PRCFC and washed platelets or red blood cells are described in supplemental methods and data.

3. RESULTS

3.1. Evaluation of VWF and fibrinogen in cryoprecipitates to support platelet adhesion under shear flow

The composition of PRCFC demonstrated enrichment for cryoprecipitated proteins compared to pathogen‐reduced plasma (Table S1). The ability of the various cryoprecipitate products and CFC to support platelet adhesion under shear flow was assessed. Human hirudinated whole blood was perfused at an arterial wall shear rate of 300 s⁻¹ through microfluidic chambers coated with Cryo‐AHF, PRCFC, LIFC, or CFC. Differential interference contrast microscopy showed stable platelet adhesion with all products (Figure 1A; Video 1). No platelets adhered to HSA, indicating that platelet adhesion to cryoprecipitates and CFC was specific (Figure 1A). The kinetics and the number of adhering platelets at 5 min were similar between Cryo‐AHF, PRCFC, and LIFC, and were reduced by 43% with CFC (Figure 1B,C). Similar experiments performed in the presence of the integrin α_IIbβ₃ inhibitor, abciximab (40 μg/mL), showed significant inhibition of platelet adhesion under all conditions, indicating a key role for the αIIbβ3/fibrinogen interaction at this shear rate (Figure 1A–C). In addition, we assessed whether β1 integrins, the other major platelet integrins, contribute to this process by blocking them with the monoclonal antibody P5D2 (10 μg/mL). Inhibition of β1 integrins did not affect platelet adhesion to Cryo‐AHF, PRCFC, LIFC, or CFC, indicating that they do not play a major role in this mechanism (Figure S1).

VIDEO 1.

Download video file ^{(13.8MB, mp4)}

Hirudinated whole blood perfusion 5 minutes, 300 s‐1, coating with HSA, cryoprecipitates or CFC.

To elucidate differences between the adhesive properties of the cryoprecipitates versus CFC, we hypothesized a role for VWF, which is present in the cryoprecipitates, but not in CFC. To test this hypothesis, we performed shear flow experiments using chambers coated with Cryo‐AHF, PRCFC, LIFC, or CFC at a wall shear rate of 300 s⁻¹ and with the addition of caplacizumab (10 μg/mL) which blocks the A1 platelet‐binding domain of VWF, preventing platelet attachment through the GPIb‐IX‐V complex. In these experiments, the cryoprecipitates were adjusted according to the fibrinogen concentration of 3 mg/mL; thus, LIFC had less VWF activity than PRCFC or Cryo‐AHF, which had equivalent VWF activity (Table S2). Based on the adjustment of the various cryoprecipitate substrates to a final fibrinogen concentration of 3 mg/mL, the respective final concentrations of VWF activity in Cryo‐AHF, PRCFC, and LIFC were: 1.48, 1.68, and 1.06 IU/mL. Caplacizumab reduced platelet adhesion to all chambers coated with cryoprecipitates, but it had no significant impact on platelet adhesion to chambers coated with CFC (Figure 1D–F). The kinetics of platelet adhesion and the quantification of the number of adhering platelets confirmed the binding capacity of the cryoprecipitates and the inhibitory effect of caplacizumab on VWF‐mediated adhesion (Figure 1E,F), resulting in 64%, 67%, and 48% reduction in the number of platelets adhering to Cryo‐AHF, PRCFC, and LIFC, respectively (Figure 1E,F). Together, these experiments indicate the relative contributions of αIIbβ3/fibrinogen and GPIb‐IX‐V/VWF‐mediated binding in supporting platelet adhesion by Cryo‐AHF, PRCFC, and LIFC under low shear flow. A‐PRT did not impair the adhesive function of fibrinogen and VWF proteins in cryoprecipitates. Notably, caplacizumab had no effect on platelet adhesion to CFC consistent with the absence of functional VWF in CFC and complete dependence on CFC fibrinogen to support platelet adhesion.

3.2. Evaluation of VWF from cryoprecipitates and CFC to support platelet adhesion under shear flow

To gain further insight into the functionality of VWF in the various cryoprecipitates, we performed flow experiments by perfusing plasma‐free blood reconstituted with cryoprecipitates or CFC, on chambers coated with a VWF‐binding peptide. This peptide allows circulating VWF adsorption, elongation, and subsequent platelet adhesion to immobilized VWF at a wall shear rate of 1500 s⁻¹. Video‐microscopy showed that numerous platelets in the presence of cryoprecipitates adhered and rolled over the surface, which confirms the typical adhesive behavior of platelets interacting with VWF through the GPIb‐IX‐V complex (Figure 2A, Video 2). Control experiments confirmed the specificity of this interaction, as no platelets adhered to HSA passivated surfaces without cryoprecipitate addition (Figure 2A). In addition, platelet adhesion was inhibited in the presence of caplacizumab, further confirming the importance of VWF contained in cryoprecipitates to support platelet adhesion under high wall shear rates (Figure 2A). In contrast, no platelets adhered to the VWF‐binding peptide when CFC was added to plasma‐free blood, confirming the absence of functional VWF in this formulation (Figure 2A). No differences in the kinetics and number of adherent platelets at 5 min were observed between Cryo‐AHF and PRCFC, indicating that A‐PRT had no major impact on the adhesive properties of VWF (Figure 2B). We observed a 35% decrease in the level of platelet adhesion between Cryo‐AHF, PRCFC, and LIFC, which is explained by lower VWF activity in LIFC of 1.06 IU/mL compared to 1.48 IU/mL and 1.68 IU/mL in Cryo‐AHF and PRCFC.

VIDEO 2.

Download video file ^{(38.3MB, mp4)}

Plasma‐free blood perfusion 3 minutes, 300 s‐1, coating with VWF binding peptide.

(Figure 2B,C; Table S2). This difference between Cryo‐AHF and PRCFC with LIFC indicates that the assay was sensitive to differences in VWF activity, and LIFC prepared from different pools of PRCFC retained sufficient activity to support platelet adhesion to the VWF binding peptide despite a lower level of VWF activity.

3.3. Evaluation of fibrinogen from cryoprecipitates to support platelet aggregation

To determine that fibrinogen in the cryoprecipitates and CFC was not qualitatively impaired, we assessed the quality of fibrinogen in the cryoprecipitates and CFC to support platelet aggregation using a Born‐type platelet aggregation assay in which we stimulated washed human platelets with 5 μM ADP. We used 30 μg/mL of fibrinogen as a threshold concentration providing a sub‐maximal response of washed platelets to 5 μM of ADP with induction of shape change and reversible platelet aggregation (Figures 3 and S2). Under this stringent condition, we observed that the platelet aggregation was similar for the various cryoprecipitates and for CFC demonstrating preservation of shape change and maximal aggregation responses of approximately 20% (Figure 3B). These results indicate that the fibrinogen of all the cryoprecipitates and CFC promoted efficient platelet aggregation, with no obvious qualitative differences between the formulations indicative of a dysfibrinogen. These results also indicated that A‐PRT and the lyophilization process had no impact on the functional quality of the fibrinogen in PRCFC and LIFC compared to Cryo‐AHF.

3.4. Evaluation of cryoprecipitates and CFC to support platelet aggregation and thrombus formation under shear flow

We assessed the ability of cryoprecipitates and CFC to support thrombus formation under higher shear flow that may be present in damaged vasculature. Microfluidic chambers were coated with type I fibrillar collagen followed by perfusion of plasma‐free blood at 3000 s⁻¹. Confocal‐video microscopy images showed the formation of large thrombi with Cryo‐AHF, PRCFC, and LIFC supplements compared with buffer, demonstrating the haemostatic efficacy of plasma proteins in cryoprecipitates to support thrombus formation. In contrast, no large thrombi were formed with CFC supplements, indicating that fibrinogen alone is not sufficient to support thrombus formation at high shear flow (Figure 4A). The kinetics of thrombus growth appeared to be similar between the three cryoprecipitates and the areas of thrombi at 5 min were not statistically different; however, PRCFC and LIFC provided significantly larger thrombi than CFC (Figure 4B,C). These results indicated that PRCFC and LIFC supported thrombus formation on collagen under high shear flow, and A‐PRT did not impair the critical functions of VWF in cryoprecipitates to support thrombus formation under high shear flow.

3.5. Evaluation of cryoprecipitates to support the aggregation of fresh and stored buffy‐coat platelets under shear flow

Because cryoprecipitates may be used in association with pathogen‐reduced platelet concentrates, we evaluated the capacity of pathogen‐reduced buffy‐coat platelet components supplemented with cryoprecipitates or CFC to support aggregation and thrombus formation after perfusion through collagen‐coated microfluidic chambers at 1500 s⁻¹. Buffy‐coat platelets were used 2, 5, and 7 days after A‐PRT and storage at 22–24°C with agitation. Confocal‐video microscopy images showed the formation of more aggregates with Cryo‐AHF, PRCFC, and LIFC supplements than with CFC at Day 2 (Figure 5A). This result was confirmed by the kinetics of the thrombus area during a 10 min perfusion, although the differences were not statistically different (Figure 5B,C). On Day 5, the thrombi area of buffy‐coat platelets was reduced compared to that observed on Day 2; and the differences in thrombus area between PRCFC and LIFC compared to CFC were significantly different (Figure 5A,D,E). On Day 7, buffy‐coat platelets exhibited reduced ability to aggregate with cryoprecipitates and CFC, but they retained significantly higher aggregation capacity with PRCFC than with CFC (Figure 5A,F,G). Interestingly, regardless of the platelet storage duration, no significant difference in thrombus areas was observed with Cryo‐AHF, PRCFC, and LIFC (Figure 5A–G). In summary, these results highlight that A‐PRT of cryoprecipitates did not affect their ability to support the aggregation of pathogen‐reduced whole blood buffy‐coat platelets under shear flow. In addition, independently of their storage time, platelets retained potential to form thrombi on collagen under high shear when perfused in the presence of cryoprecipitates.

4. DISCUSSION

In this study, we evaluated the impact of A‐PRT on soluble proteins in cryoprecipitates and CFC to support platelet hemostatic functions using an in vitro microfluidic model to simulate in vivo vascular conditions. Although the microfluidic model is not the equivalent of in vivo vasculature, it allows control of experimental variables to characterize platelet interactions with relevant constituents such as VWF and fibrinogen. We observed that VWF and fibrinogen from A‐PRT cryoprecipitates supported efficient platelet adhesion and aggregation under low and elevated shear flow. Notably, we observed that cryoprecipitates support greater platelet adhesion and aggregation than CFC, mainly due to the presence of functional VWF.

To evaluate whether various cryoprecipitates are able to support the initial stage of hemostasis, we assessed platelet adhesion to immobilized cryoprecipitate proteins under physiological arterial shear at 300 s⁻¹. ²⁶ All products demonstrated stable platelet adhesion, and similar levels of adhesion were observed with Cryo‐AHF, PRCFC, and LIFC. Inhibition of integrin αIIbβ3 interaction with fibrinogen, fibronectin and VWF abolished platelet adhesion, indicating that at least one of these proteins is necessary for efficient platelet adhesion under shear flow. The number of platelets adhering to cryoprecipitates was two times greater compared to CFC for a similar fibrinogen concentration. As CFC contains only fibrinogen, this suggested the involvement of a factor other than fibrinogen in Cryo‐AHF, PRCFC, and LIFC, or lesser efficacy of fibrinogen in CFC. The aggregation assay responses with low‐dose ADP showed similar platelet aggregation between all products, excluding lower efficacy of CFC fibrinogen. The inhibition of β1 integrin with the P5D2 monoclonal antibody did not affect platelet adhesion on Cryo‐AHF, PRCFC, and LIFC. This suggests that integrin α5β1 and fibronectin are not essential to support platelet adhesion. We evaluated the involvement of another critical adhesive protein, VWF, to explore the differences in platelet adhesion. Caplacizumab, an antagonist of VWF, reduced the number of adherent platelets with Cryo‐AHF, PRCFC and LIFC to the level observed with CFC, indicating that cryoprecipitates support more platelet adhesion compared to CFC at 300 s⁻¹ due to the presence of VWF.

A key step during hemostasis is the binding of VWF to collagen, enabling it to unfold, slowing down platelets with localization to the injured site. ¹⁷ Perfusion of plasma‐free blood, supplemented with cryoprecipitates or CFC, on a VWF binding peptide promoted transient platelet adhesion, typical of VWF binding for all cryoprecipitates, but no adhesion was observed with CFC. This indicates that the VWF in cryoprecipitates bound and unfolded under flow, supporting efficient platelet attachment. We observed that Cryo‐AHF and PRCFC exhibited VWF activity, consistent with observations that VWF from PRCFC or Cryo‐AHF enable equivalent responses in the RIPA assay. ²⁷ In contrast, LIFC showed reduced adhesion compared to Cryo‐AHF and PRCFC, likely due to a lower concentration of VWF in LIFC used for these experiments, as conditions were normalized for fibrinogen and not VWF content.

Recently, Thomas et al. reported a difference in platelet aggregation between PR‐cryoprecipitates versus conventional cryoprecipitates in a different microfluidic model. ²⁸ They proposed that the reduction in platelet aggregation might be attributed to a defect in VWF unfolding. As their flow assay is not specific for VWF‐platelet interactions, but instead used an integrated in vitro hemostasis model with perfusion of recalcified citrated whole blood over collagen, the defect they observed with PRCFC could arise from VWF‐independent mechanisms such as GPVI‐collagen, α2β1‐collagen, or αIIbβ3‐fibrinogen platelet interactions. This hypothesis is in line with our observations showing no defect in flow‐mediated platelet adhesion and aggregation with A‐PRT cryoprecipitates and conventional cryoprecipitate in a model in which platelet attachment to the surface exclusively relies on VWF. Importantly, we used a specific VWF‐mediated adhesion assay in which reconstituted whole blood perfused over a VWF‐binding peptide measured capture of flowing VWF, followed by unfolding and platelet recruitment. We did not observe any defect in platelet attachment, indicating that it is unlikely that A‐PRT impairs VWF unfolding.

Plasma‐free hirudin anticoagulated blood perfused over immobilized collagen under arterial shear flow showed that all cryoprecipitates facilitated larger thrombus formation than CFC, suggesting improved efficacy. This is consistent with data indicating that VWF is required at high shear flow to support thrombus formation. ²⁹ During massive hemorrhage, cryoprecipitate can be transfused in combination with platelet concentrates. ³⁰ However, the storage of platelet concentrates leads to a reduction of platelet activity over time. ³¹ We observed that long‐stored buffy‐coat platelet components in plasma‐free blood demonstrated less aggregation to collagen. However, after 2, 5, or 7 days of platelet storage, aggregation was improved with supplemental Cryo‐AHF, PRCFC, or LIFC, but not with CFC.

Our study used assays that are informative about critical platelet–cryoprecipitate interactions to complement recent studies on the impact of A‐PRT on cryoprecipitates which described reduced fibrinogen, VWF, Factor VIII, and Factor XIII content. ⁶ , ¹⁴ , ¹⁶ We observed retention of critical platelet hemostatic functions with A‐PRT cryoprecipitates, and our data complement observations by Kamyszek et al., showing that A‐PRT had no impact on thrombin generation. ¹⁵

A major reason that CFC replaced cryoprecipitates was lack of pathogen inactivation methods for cryoprecipitates. A‐PRT mitigates the risk of bacterial contamination and allows extension of post‐thaw storage of PRCFC to 5 days. In this study, we observed that PRCFC and LIFC provided better hemostasis support compared with CFC in arterial shear flow conditions. PRCFC was a more physiologic fibrinogen supplement than CFC.

While the number of replicates used in our study due to assay complexity is a potential limitation, we observed consistent biological results with several different but complementary assays. Another limitation of our study was that the microfluidic experiments were normalized to fibrinogen concentration and not to VWF. However, we observed that there was sufficient VWF remaining in all the cryoprecipitates to support platelet function.

In future studies, it will be interesting to evaluate the effect on PRCFC manufactured with a recently developed LED light device instead of the current UVA light device. A recent publication describing the use of the LED light device for manufacturing platelet concentrates demonstrated comparable performance. ³¹ As pathogen inactivation allows PRCFC to be used for up to 5 days after thawing, ⁶ it would also be interesting to assess the ability of cryoprecipitates to improve clinical platelet function after 5 days of post‐thaw storage. To supplement the limitation of the current study with in vitro models, it could be informative to evaluate the effectiveness of PRCFC and LIFC in restoring in vivo hemostasis without increased thrombotic risk using fibrinogen or VWF deficient animal models. In conclusion, our study provides evidence that PRCFC retains VWF and fibrinogen platelet hemostatic interactions in vitro under simulated physiologic and pathologic vessel wall shear rates supporting use in treatment of massive hemorrhage.

AUTHOR CONTRIBUTIONS

Florian Tupin and Pierre Mangin designed the assays. Clarisse Mouriaux and Florian Tupin performed the microfluidic assays. Michelle Gatmaitan assayed coagulation factor content of study articles. Subra Yegneswaran directed the production of study articles. Kaja Kaastrup conducted assays for FDA registration studies for PRCFC. Laurence Corash and Pierre Mangin contributed to the design of the studies. All the authors participated in the writing and editing of the manuscript.

FUNDING INFORMATION

This study was supported by a contract from CERUS Corporation (Concord, CA), and funding from INSERM, EFS, and ARMESA (Association de Recherche et Développement en Médecine et Santé Publique). FT was supported by a fellowship from Region Grand Est and ARMESA.

CONFLICT OF INTEREST STATEMENT

Laurence Corash, Kaja Kaastrup, Michelle Gatmaitan, and Subra Yegneswaran are employees of Cerus Corporation and beneficial owners of Cerus equity. Pathogen Reduced Plasma, Cryoprecipitate Reduced is a product of Cerus Corporation marketed in the U.S. Florian Tupin, Clarisse Mouriaux, and Pierre H. Mangin have no conflicts of interest to declare. The study was supported by funds from Cerus.

Supporting information

Table S1. Composition of pathogen‐reduced cryoprecipitated fibrinogen complex (PRCFC) compared with pathogen‐reduced plasma.

Table S2. Concentration of fibrinogen and VWF activity for the three different cryoprecipitates used in microfluidic experiments.

Figure S1. Evaluation of β1 integrin impact on platelet adhesion to cryoprecipitates and CFC under flow.

Figure S2. Platelet aggregation to ADP with varying fibrinogen concentrations.

Figure S3. Preparation of PRCFC.

Data S1. Supporting information.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.