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Published in final edited form as: J Thromb Haemost. 2023 Dec 23;22(4):1016–1023. doi: 10.1016/j.jtha.2023.12.017

Dual antithrombotic therapy dose-dependently alters hemostatic plug structure and function

Christopher D Mansi *, Jenna R Severa *, Joseph N Wilhelm *, Tanya T Marar *, Meghan E Roberts *, Xuefei Zhao *, Timothy J Stalker *
PMCID: PMC10960666  NIHMSID: NIHMS1959991  PMID: 38142847

Abstract

Background:

Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and anti-platelet therapeutics are combined. Prior studies have shown different effects of anti-platelet versus anticoagulant drugs on the structure and function of hemostatic plugs in vivo.

Objectives:

We examined whether dual antithrombotic treatment consisting of combined anti-platelet and anticoagulant therapeutics alters hemostatic plug structure and function differently than treatment with either therapeutic alone.

Methods:

Mice were treated with the P2Y12 antagonist clopidogrel and the Factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy and bleeding times recorded.

Results:

Mice treated with clopidogrel alone had a decrease in platelet accumulation at the site of injury with prolonged bleeding times only at the highest doses of clopidogrel used. Mice given rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban had platelet and fibrin accumulation that was similar to either drug given alone, however, dual anti-thrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation.

Conclusions:

Combined administration of anti-platelet and anticoagulant therapeutics exacerbates bleeding as compared to either drug alone, potentially via combined loss of both ADP- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy.

Keywords: Hemostasis, Purinergic P2Y12 receptor antagonist, Factor Xa inhibitors, Multiphoton fluorescence microscopy, Anticoagulants

Introduction

Given the contribution of both thrombin and platelets to thrombosis in its various manifestations, it has been postulated that dual antithrombotic treatment (DAT) with both an anticoagulant and anti-platelet agent would have improved efficacy compared to treatment targeting either coagulation or platelets in isolation (reviewed in [1, 2]). Recent clinical studies examining the efficacy of factor Xa inhibition in combination with anti-platelet therapy in the setting of coronary arterial disease (CAD) and peripheral arterial disease have shown a benefit of DAT in reducing major adverse cardiovascular events [3–5]. Further, additional patient populations exist where the choice of anti-thrombotic strategy is unclear. Patients with left ventricular assist devices (LVADs), myeloproliferative neoplasms (e.g. polycythemia vera), left ventricular thrombosis associated with myocardial infarction, and co-morbities such as CAD in addition to atrial fibrillation may all receive both anticoagulants and anti-platelet agents [6–9]. In each case, balancing antithrombotic effect with bleeding risk is a significant clinical challenge. With regard to bleeding risk, a better understanding of how anticoagulants and anti-platelet agents, alone or in combination, affect hemostatic plug architecture to cause bleeding may help inform clinical decision making.

Platelet recruitment, platelet activation, thrombin generation, and fibrin formation are all tightly regulated in time and space to achieve an optimal hemostatic response. Prior studies have demonstrated that anti-platelet and anticoagulant therapeutics have distinct effects on hemostatic plug structure. Anti-platelet agents such as P2Y12 antagonists and aspirin attenuate the accumulation of minimally activated platelets on the luminal surface of a platelet plug without substantially impairing the accumulation of highly activated platelets or fibrin formation [10, 11]. Inhibition of thrombin activity instead impairs robust platelet activation as well as fibrin formation [12–14]. Due to the non-redundant contribution of these molecular pathways to hemostasis, we hypothesized that combined inhibition of both thrombin generation and platelet P2Y12 signaling would disrupt hemostatic plug structure and exacerbate bleeding to a greater extent than when either pathway is targeted alone. To test this hypothesis, we utilized a mouse large vessel injury model coupled with multiphoton imaging to determine the consequences of DAT on hemostatic plug structure and function in vivo.

Methods

Materials

Male C57BL/6J mice (Jackson Laboratories) ages 8–12 weeks were used for all studies. Animal studies were approved by the IACUC of Thomas Jefferson University. Alexa 647 labelled fibrinogen was purchased from ThermoFisher Scientific (Waltham, MA). Anti-CD41 clone MWReg30, F(ab)2 was purchased from BD Biosciences (San Jose, CA) and was labeled with Alexa 568 using the monoclonal antibody labeling kit from ThermoFisher according to the manfacturer’s instructions. Fluorescent reagents were administered via retro orbital injection immediately before surgery. Rivaroxaban (Sellekchem, Houston, TX) was dissolved in dimethyl sulfoxide (DMSO) and suspended in polyethylene glycol (PEG) as previously described [15], and administered via direct infusion into the left jugular vein 5 minutes prior to puncture injury. Clopidogrel bisulfate (Sellekchem) was dissolved in water and administered via oral gavage 3 hours prior to surgery.

Jugular vein puncture injury model

Mouse jugular vein puncture, imaging, and analysis were performed as described previously [11, 16]. The time to cessation of bleeding as visualized through a dissecting microscope was recorded as the bleeding time [11]. Images were acquired using a 25x (1.0 NA) water dipping objective with a Leica TCS SP8 multiphoton confocal microscope at the University of Pennsylvania. 3D reconstructions were analyzed using Imaris image analysis software (Oxford Instruments, Concord, MA). Volumetric measurements were acquired for platelets and sum fluorescence intensity for fibrin, as previously described [11]. As CD41 expression is on average consistent among individual platelets, both platelet volume and CD41 sum fluorescence intensity are good indicators of total platelet accumulation. Platelet volume is reported here as we consider the units of μm3 more meaningful than the arbitrary units associated with fluorescence intensity. Fibrin may vary in intensity within a given 3D volume depending on the extent and structure of fibrin fiber formation. Accordingly, fibrin sum intensity is reported as a measure of total fibrin deposition.

Statistics

Statistical analysis and graphs were produced using Prism 6.0 software (GraphPad). Platelet and fibrin accumulation were compared among experimental groups using the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons. For post hoc testing, each experimental group was compared with the control (vehicle) group, and comparisons were also made among dual antithrombotic therapy groups vs single antithrombotic agents at equivalent doses. P values are shown for all comparisons where p<0.05; all other comparisons were not statistically significant (p>0.05). Bleeding times were compared using Kaplan-Meier analysis (Log-rank test) [17].

Results and Discussion

Impact of single or dual antithrombotic therapy on platelet accumulation

The Factor Xa inhibitor, rivaroxaban, was used as an anticoagulant in doses ranging from 0.1–1 mg/kg. This range was based on a dose-dependent prolongation of the prothrombin time of plasma isolated from mice given rivaroxaban intravenously (Figure 1A). The low and high doses chosen for subsequent studies (0.1 and 1 mg/kg) approximate low (2.5 mg) and high (20 mg) doses of rivaroxaban administered to humans based on their effect on prothrombin time[18]. Clopidrogrel was chosen as an anti-platelet agent since it remains the most widely prescribed P2Y12 antagonist in clinical use [19]. It is also less potent than ticagrelor and has reduced risk of spontaneous bleeding [20]. The dose range chosen was based on in vitro studies in which platelet rich plasma isolated from clopidogrel treated mice exhibited a dose-dependent decrease in ADP-induced platelet aggregation. The low and high doses used (5 and 25 mg/kg) resulted in an approximately 40 and 60 percent decrease in ADP-induced platelet aggregation, respectively (Figure 1B). The jugular vein of mice treated with either vehicle, rivaroxaban alone, clopidogrel alone, or combined clopidogrel and rivaroxaban was punctured using a 30-gauge (300 μm diameter) needle, and the time to achieve hemostasis was recorded. After 5 minutes the mouse was perfusion fixed, and the jugular vein excised for imaging using multiphoton microscopy.

Figure 1: Dose effects of rivaroxaban and clopidogrel in mice.

Figure 1:

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Mice were administered vehicle or clopidogrel (5–25 mg/kg) via oral gavage three hours prior to blood collection. Mice were administered vehicle or rivaroxaban (0.1–1 mg/kg) via intravenous infusion five minutes prior to blood collection. A) Prothrombin time measurement. Values are mean±SEM; n=3 mice per group. B) PRP was isolated and aggregation was induced using 10 μM ADP. Values are mean±SEM; n=4–10 mice per group.

In vehicle treated mice, robust platelet accumulation at the injury site resulted in the formation of a stable hemostatic plug. The hemostatic plug was composed primarily of platelets that formed a mound over the injury site in the intraluminal compartment that extended through the hole in the vessel wall to the extraluminal compartment (Figure 2A). On the extraluminal side, platelets formed small aggregates spread along the surface of the blood vessel around the periphery of the injury site (Figure 2B). Treatment of mice with rivaroxaban alone resulted in a trend towards decreased platelet accumulation within the intraluminal compartment that was not statistically significant (Figure 2C, blue columns; p>0.99, p=0.19 for 0.1 mg/kg and 1.0 mg/kg vs. vehicle, respectively). Extraluminal platelet accumulation was similar in vehicle and rivaroxaban treated mice (Figure 2D).

Figure 2: Platelet accumulation following mouse jugular vein puncture injury in the setting of single or dual antithrombotic treatment.

Figure 2:

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A-B) Representative images show platelet deposition five minutes post-injury on the intraluminal (A) and extraluminal (B) sides of the punctured vessel wall. Images are 3-dimensional reconstructions of optical sections acquired using multiphoton microscopy. Platelets are shown in magenta and vessel wall collagen in white (second harmonic imaging). Scale bars are 80 μm. C-D) Graphs show total platelet volume on the intraluminal (C) and extraluminal (D) sides of the punctured vessel wall. Bars indicate median with interquartile range; dots show data points from individual mice. Gray dots indicate mice that did not stop bleeding within the 5 minute observation period. N=18 for vehicle; n=6 and 11 for 0.1 and 1 mg/kg rivaroxaban alone; n=8 and 7 for 5 mg/kg clopidogrel and 25 mg/kg clopidogrel alone; n=10 for 0.1 mg/kg rivaroxaban + 5 mg/kg clopidogrel; n=11 for 1 mg/kg rivaroxaban + 5 mg/kg clopidogrel. Statistics were performed using the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons. Only p-values <0.05 are shown; all other comparisons were not significant (p>0.05).

Mice treated with the high dose of clopidogrel (25 mg/kg) had significantly impaired platelet accumulation, particularly on the intraluminal side of the hemostatic plugs (p<0.01, vehicle vs. 25 mg/kg clopidogrel, Figure 2A, C, red columns), consistent with prior reports examining the impact of P2Y12 receptor inhibition in this hemostasis model [11]. Mice treated with the low dose of clopidogrel (5 mg/Kg) formed stable hemostatic plugs that showed no changes in either intraluminal or extraluminal platelet accumulation (Figure 2C–D, red columns), despite the inhibitory effect of this dose of clopidogrel on platelet aggregation observed in vitro.

Dual antithrombotic treatment consisted of either the low or high dose of rivaroxaban (0.1 and 1 mg/kg) along with the low dose of clopidogrel (5 mg/kg). Only the low dose of clopidogrel was used for DAT studies as the higher dose of clopidogrel tested consistently resulted in failure to achieve hemostasis in the puncture injury model employed here (bleeding data discussed in more detail below). Mice treated with the combination of low dose clopidogrel and either low or high dose rivaroxaban did not show any significant changes in platelet accumulation on average when compared to vehicle or each therapy alone (Figure 2A–D, purple columns). Notably, the mice receiving clopidogrel and high dose rivaroxaban that had the lowest intraluminal platelet accumulation also had an impaired hemostatic response (Figure 2C, gray dots; discussed in more detail below).

Impact of single or dual antithrombotic therapy on fibrin deposition

Fibrin deposition following puncture injury is primarily localized extraluminaly, spread across the outer surface of the injured blood vessel and lining the circumference of the injury site itself (Figure 3A–B). Minimal fibrin accumulation is observed within the intraluminal compartment, although the small amount of fibrin found there in vehicle treated mice was significantly attenuated in mice treated with either dose of rivaroxaban alone (Figure 3C, blue columns). Rivaroxaban treatment significantly reduced extraluminal fibrin formation at the highest dose used (p<0.001, vehicle vs. 1 mg/kg, Figure 3D, blue columns). Treatment of mice with clopidogrel alone had no impact on fibrin formation in either the intraluminal or extraluminal compartments (Figure 3C–D, red columns).

Figure 3: Fibrin formation following mouse jugular vein puncture injury in the setting of single or dual antithrombotic treatment.

Figure 3:

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A-B) Representative images show platelet and fibrin deposition five minutes post-injury on the extraluminal side of the punctured vessel wall. Images in B) are the same samples shown in A) with the platelets digitally removed to better visualize fibrin deposition. Images are 3-dimensional reconstructions of optical sections acquired using multiphoton microscopy. Platelets are shown in magenta, fibrin in green and vessel wall collagen in white (second harmonic imaging; collagen signal in some images is obscured by platelet accumulation). Injury site is shown by the yellow dotted line. Scale bars are 80 μm. C-D) Graphs show fibrin sum intensity on the intraluminal (C) and extraluminal (D) sides of the punctured vessel wall. Bars indicate median with interquartile range; dots show data points from individual mice. Gray dots indicate mice that did not stop bleeding within the 5 minute observation period. N=18 for vehicle; n=6 and 11 for 0.1 and 1 mg/kg rivaroxaban alone; n=8 for 5 mg/kg clopidogrel; n=7 for 25 mg/kg clopidogrel; n=10 for 0.1 mg/kg rivaroxaban + 5 mg/kg clopidogrel; n=11 for 1 mg/kg rivaroxaban + 5 mg/kg clopidogrel. Statistics were performed using the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons. Only p-values <0.05 are shown; all other comparisons were not significant (p>0.05).

Mice treated with both clopidogrel and rivaroxaban displayed a decrease in fibrin formation (Figure 3C–D, p<0.001 vehicle vs. clopidogrel + 1 mg/kg rivaroxaban, purple columns). The extent of fibrin inhibition was similar in the face of dual antithrombotic treatment as compared to treatment with either dose of rivaroxaban alone. These results demonstrate that P2Y12 antagonism does not further reduce fibrin formation in the context of Factor Xa inhibition.

Impact of single or dual antithrombotic therapy on bleeding

A competent hemostatic plug formed in 13 out of 14 vehicle treated mice with an average bleeding time of 111.5±8.7 seconds (mean±SEM, Figure 4A–B). Despite the substantial reduction in fibrin formation described above, mice treated with either low or high dose rivaroxaban alone had no significant difference in either the number of mice forming competent hemostatic plugs, or the bleeding time as compared to vehicle treated mice (Figure 4A–B, blue lines/columns). In contrast, P2Y12 antagonism with high dose clopidogrel resulted in a significant hemostatic deficit, with only 1 out of 7 mice forming a competent hemostatic plug (p=0.0024, vehicle vs. 25 mg/kg clopidogrel, Figure 4A–B, red lines/columns). Treatment with the low dose of clopidogrel alone did not result in excess bleeding as compared to vehicle controls (p=0.79, Figure 4A–B).

Figure 4: Bleeding times following mouse jugular vein puncture injury in the setting of single or dual antithrombotic treatment.

Figure 4:

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A) Graph shows time to cessation of bleeding following jugular vein puncture injury. Values are mean±SEM; dots show data points from individual mice. Mice that had not stopped bleeding at 300 s when the experiment was terminated are noted as >300 s (gray dots). B) Graph shows Kaplan-Meier analysis of bleeding time data. N=18 for vehicle; n=6 and 11 for 0.1 and 1 mg/kg rivaroxaban alone; n=8 for 5 mg/kg clopidogrel; n=7 for 25mg/kg clopidogrel; n=10 for 0.1 mg/kg rivaroxaban + 5 mg/kg clopidogrel; n=11 for 1 mg/kg rivaroxaban + 5 mg/kg clopidogrel. Statistics were performed using the log-rank test (each survival curve was compared to the vehicle group). Only p-values <0.05 are shown; all other comparisons were not significant (p>0.05).

Mice treated with clopidogrel plus the low dose of rivaroxaban displayed a trend towards enhanced bleeding that was not statistically significant (p=0.76, Figure 4A–B, light purple lines/columns). Five out of 11 mice receiving clopidogrel plus 1 mg/kg rivaroxaban failed to achieve hemostasis within the 5-minute observation period, with substantially more bleeding than vehicle controls or mice treated with either of these drugs in isolation (p=0.011, vehicle vs. clopidogrel + 1 mg/kg rivaroxaban, Figure 4A–B, dark purple lines/columns). As noted earlier, mice in this group that failed to achieve hemostasis also had low platelet accumulation (Figure 1C, gray dots). These results show that dual antithrombotic therapy has the potential to exacerbate bleeding as compared to mono-pathway antithrombotic approaches, possibly as a result of intensified platelet inhibition in the context of P2Y12 antagonism and attenuated thrombin mediated platelet activation.

Taken together, the results of the current study show that combined inhibition of thrombin generation and platelet P2Y12 signaling impairs the hemostatic response to a greater extent than targeting either pathway alone. As in vitro studies showed that P2Y12 antagonism does not alter coagulation, and Factor Xa inhibition does not impair platelet activation (Figure 1), it is likely that the effects of these therapeutics on hemostasis in vivo is related to direct action on their intended targets. The finding that fibrin formation was no different in mice treated with a Xa antagonist alone or in combination with a P2Y12 antagonist suggests that impaired fibrin formation itself is unlikely to explain the increased bleeding observed in the setting of DAT. Instead, we found that platelet accumulation was lowest in mice that failed to achieve hemostasis, suggesting that excess bleeding results from attenuated platelet activation. These results are consistent with thrombin and platelet ADP signaling contributing separate (perhaps overlapping) functional effects during hemostatic plug formation. In addition to fibrin formation, thrombin contributes in a meaningful way to platelet accumulation/activation via PAR receptor activation [21–23]. From a spatiotemporal point of view, thrombin generation during early hemostasis is primarily extravascular, due to localization of tissue factor expressing cells and the impact of physical forces such as the convective effects of extravasating flow [16]. Accordingly, thrombin-mediated platelet activation would likely occur in the extraluminal compartment. Yet, platelet accumulation defects in the context of attenuated thrombin generation are observed intraluminally, similar to the effect of P2Y12 antagonism [11, 14, 24]. One possible explanation for these results is that impaired thrombin-mediated platelet activation extraluminally results in decreased platelet dense granule secretion and ADP release impacting intraluminal platelet recruitment. Alternatively, spatial propagation of thrombin generation/activity within the platelet plug may be required for direct PAR-mediated activation of platelets within the intraluminal compartment to maintain stable platelet adhesion. The results of the current study cannot distinguish between these possibilities and they are not mutually exclusive.

Multiple limitations of the current study should be noted. The mouse jugular vein puncture injury model used here represents a single physiologic context. It is possible that differences in blood flow, pressure, vessel wall biology, and injury size in different blood vessels and vascular beds will alter the relative importance of ADP versus thrombin mediated platelet activation for the cessation of bleeding. Also, the current study focused on the initial formation of hemostatic plugs. Future studies examining the impact of DAT on hemostatic plug stability over longer time periods would be worthwhile. Finally, while the current study examined bleeding in the context of dual antithrombotic therapy, antithrombotic efficacy of DAT at equivalent doses was not evaluated. Nonetheless, the results highlight the complex interplay between coagulation and platelets in the formation of a hemostatic plug and provide additional evidence regarding potential risks associated with dual antithrombotic therapy.

Acknowledgements

Special thanks to Dr. Andrea Stout of the CDB Microscopy Core at the University of Pennsylvania for assistance with multiphoton imaging, and to Drs. Francis Ayombil and Rodney Camire at Children’s Hospital of Philadelphia for assistance with prothrombin time assays. The authors gratefully acknowledge research funding from the National Heart, Lung and Blood Institute (P01-HL139420 and P01-HL146373) and the American Heart Association (19TPA34880016).

Footnotes

Conflict of interest disclosures

The authors have no conflicting financial interests.

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