Measurement of plasma and platelet tissue factor pathway inhibitor, factor V and Protein S in people with haemophilia

Abstract

Introduction:

Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B.

Aim:

To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding.

Methods:

Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors.

Results:

There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S.

Conclusion:

Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.

1 |. INTRODUCTION

Haemophilia A and haemophilia B are bleeding disorders caused by insufficient activity of the plasma blood clotting proteins, factor VIII (FVIII) or factor IX (FIX), respectively. Inherited forms of haemophilia occur in 1:5000 male births.^1,2 Acquired haemophilia is a rare disorder that typically results from the production of autoantibodies directed against FVIII.³ Severe forms of inherited and acquired haemophilia are associated with spontaneous bleeding into joints and soft tissues.⁴ The bleeding can be treated with intravenous infusion of either the missing clotting factor or a bypassing agent, such as activated factor VII (FVIIa) or activated prothrombin complex concentrates.⁵ The need for frequent intravenous infusions negatively affects the quality of life for haemophilia patients and compliance with treatment.⁴ Thus, the development of effective therapies requiring less frequent and non-intravenous administration is desired. Emicizumab, a bivalent antibody that circumvents the requirement for FVIII by binding factors IX and X, is the only clinically available haemophilia therapy that can be delivered subcutaneously.⁶ Several others are currently in the development that block the activity of tissue factor pathway inhibitor (TFPI).^7–9

TFPI is an anticoagulant protein produced in two isoforms, TFPIα and TFPIβ (reviewed in¹⁰). Both directly inhibit factor Xa (FXa) and, in a FXa-dependent manner, inhibit FVIIa within the tissue factor (TF)-FVIIa complex. Furthermore, TFPIα binds a form of activated factor V (FV) that retains the acidic region within its B domain.¹¹ Through this interaction, TFPIα exerts isoform-specific anticoagulant activity by slowing FV activation,¹² and inhibiting early forms of prothrombinase.¹¹ The clinical importance of this interaction is high-lighted in patients who express FV East Texas¹³ or FV Amsterdam,¹⁴ variant forms of FV that tightly associate with plasma TFPIα. These patients have 10- to 20-fold increased plasma TFPIα that produces a moderately severe bleeding diathesis. Protein S also binds to TFPIα.^15,16 This interaction, mediated by the third Kunitz domain of TFPIα^17,18 and the laminin-G-type domains within the SHBG region of Protein S,¹⁹ enhances the inhibition of FXa approximately 10-fold.¹⁶ FV, particularly the FV2 isoform, also acts as a cofactor to enhance inhibition of FXa by TFPI.^20,21 This cofactor activity requires the presence of Protein S.²²

In the presence of TFPI anticoagulant activity, normal haemostasis requires an amplification of the procoagulant response via FVIII and FIX.²³ Patients with haemophilia cannot amplify coagulation in this manner and suffer from severe bleeding. However, blocking TFPI activity in plasma from haemophilia patients in vitro restores normal thrombin generation and clot formation following addition of dilute TF.²⁴ Similarly, blocking TFPI activity restores haemostasis in mouse,²⁵ rabbit,²⁶ dog²⁷ and monkey²⁸ models of haemophilia. In a recent phase 1b clinical trial, the administration of concizumab, an antibody directed against both TFPI isoforms, to normal or individuals with haemophilia, resulted in elevated plasma prothrombin fragment 1 + 2 and D-dimer concentrations in both cohorts.²⁹ These results demonstrate that this therapy increases in vivo thrombin generation and advocates further investigation into its use for the treatment of haemophilia.

The majority of plasma TFPI is C-terminally truncated, bound to lipoproteins³⁰ and has decreased anticoagulant activity compared with plasma TFPIα.³¹ Plasma TFPIα accounts for approximately 20% of total plasma TFPI³² and circulates bound to FV³³ and/or PS.³⁴ Approximately 10% of TFPI present in whole blood is concentrated within platelets.³⁵ Platelet TFPI, which is exclusively TFPIα, is secreted from activated platelets as a soluble protein,^35,36 with a portion also localizing to the activated platelet surface.³⁶ In vivo thrombosis and bleeding models have demonstrated that platelet TFPI modulates clot structure in wild-type mice and bleeding severity in FVIII-deficient mice.^25,37 These murine experiments, along with the biochemical data uncovering the inhibition of prothrombinase by TFPIα,¹¹ implicate the inhibition of platelet TFPI as an important therapeutic strategy for anti-TFPI haemophilia therapies.

We previously measured the plasma and platelet concentrations of TFPI, FV and PS in a cohort of 435 blood donors to gain a better understanding of how these factors correlate with each other and how they vary by demographic characteristics in a healthy population.³² This study revealed platelet TFPI correlates poorly with plasma TFPI, as well as with platelet FV and PS. Racial and ethnic differences in levels of the various factors were observed, but for most factors, these differences were less than inter-individual differences. However, it is not clear whether there are differences in plasma and platelet TFPI, FV and PS between healthy blood donors and people with haemophilia. Indeed, there is evidence to suggest that plasma TFPI concentration is decreased in patients with haemophilia A³³ and FVIII-deficient plasma.³⁴ Furthermore, individuals with haemophilia B may have lower plasma TFPIα concentrations than those with haemophilia A.³⁸ To gain further insight into plasma and platelet TFPI, FV and PS in people with haemophilia, we measured the concentration of these factors in 91 individuals with haemophilia A and haemophilia B.

2 |. METHODS

2.1 |. Ethics approval

Human ethics approval was granted by the Institutional Review Boards of the Blood Center of Wisconsin and the Children’s Hospital of Wisconsin. Written, informed consent for enrolment in this study was obtained from each participant before sample collection.

2.2 |. Blood collection and processing

A 5 mL citrated whole blood sample was collected from study participants during a phlebotomy visit scheduled for routine clinical care. It was centrifuged [150 × g, 15 minutes, room temperature (RT)] and the platelet-rich plasma (PRP) collected. The remaining blood sample was centrifuged (2500 × g, 20 minutes, RT) and the plasma isolated and stored (−80°C). An equal volume of BSGC buffer (129 mmol/L NaCl, 13.6 mmol/L Na₃citrate, 11.1 mmol/L glucose, 1.6 mmol/L KH₂PO₄, 8.6 mmol/L NaH₂PO₄, 100 ng/mL prostaglandin E-1, pH 7.3) was added to the PRP. Platelets were pelleted by centrifugation (650 × g, 10 minutes, RT), washed (2× with BSGC buffer), counted and placed in lysis buffer [20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% Triton X-100, 1 μg/mL E-64 (N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine), 1 mmol/L phenylmethanesulfonylfluoride, pH 7.5] at 1 × 10⁹ platelets/L. The lysate was clarified by centrifugation (14 000 × g, 10 minutes, 4°C) and the supernatant stored at −80°C. The concentration of each platelet analyte was normalized to total protein content of the lysate as determined by BCA assay.

2.3 |. Assays for Total TFPI, TFPIα, Total Protein S and Factor V

Total TFPI and TFPIα-specific bead-based proximity assays were performed as previously described.³² Total PS and FV ELISAs (Enzyme Research Laboratories) were performed according to manufacturers’ instructions. Testing of plasma proteins for blood donors and people with haemophilia was performed using the same methods, but at different times. For all assays, a pooled normal plasma or pooled platelet lysate, produced from 20 healthy males, was included in each assay run to monitor for assay drift and shift. Results obtained with the pooled sample were compared to a Levy-Jennings plot produced for each assay, and all results from an assay run were discarded and rerun if the result of the pooled sample was out of range.

2.4 |. Statistical Analysis

All data were log-transformed (natural logarithm) prior to statistical analysis, and differences on the log scale were back-transformed (by the exp function) to the original scale as ratios (or fold). Data were analysed by a series of models. The initial model included the following factors as explanatory variables: age at enrolment as a continuous regression parameter in all models; and haemophilia type (A or B) or haemophilia severity (mild/moderate or severe) as categorical factors. The initial model further included those interaction terms, which were identifiable from data. The initial model was reduced by removal of non-significant terms (respecting the model hierarchy). Equations used to calculate adjusted values are as previously published.³² The correlation between the seven parameters was estimated by the restricted maximum-likelihood method using the log-transformed results. Calculations were performed using SAS ® JMP version 12.0.1.

3 |. RESULTS

3.1 |. Study population

This study enrolled 91 people with congenital haemophilia (Table 1). There were 73 with haemophilia A: 46 severe, 12 moderate and 15 mild; and 18 with haemophilia B: 11 severe, 2 moderate and 5 mild. The distribution of haemophilia type (A: 80.2%; B: 19.8%) and severity (severe: 62.6%; moderate: 15.4%; mild: 22.0%) is similar to that observed in other regions of the United States.² Haemophilia severity was assigned according to the classification scheme of Biggs and MacFarlane.^39,40 The haemophilia cohort included five sets of related individuals—two brothers with mild haemophilia A, two sets of two brothers with severe haemophilia A, two brothers with severe haemophilia B, and a father and son with severe haemophilia B. The average ages of the haemophilia A, haemophilia B and blood donor populations were 25.6 ± 19.5, 22.2 ± 17.9 and 43.3 ± 18.6 (years; average ± SD), respectively. Other demographic data are presented in Table 1. Those with mild or moderate haemophilia were analysed together to improve statistical power.

TABLE 1.

Demographics of the study population, stratified by haemophilia type and severity

Population	Haemophilia A		Haemophilia B		Blood Donors
Haemophilia Severity	Mild/Moderate	Severe	Mild/Moderate	Severe	N/A
Age [Average (Range; n)]
<18 y old	9.5 (4–17; 13)	9.0 (2–17; 19)	13.0 (10–16; 2)	8.9 (1–17; 8)	7.6 (3–11; 9)
≥18 old	50.2 (18–73; 14)	31.7 (18–59; 27)	42.4 (21–61; 5)	30.3 (25–35; 3)	45.1 (18–87; 215)
Ethnicity
Caucasian	16	40	7	3	169
African American	2	3	0	4	27
African American/Asian	0	2	0	0	0
Asian	2	0	0	2	16
Hispanic	7	1	0	2	0
Co-morbidities Present
HIV	0	4	0	0	0
HCV	4	6	1	0	0
Inhibitor Present	1	3	0	2	N/A
Treatment
On-demand	27	14	6	3	N/A
Episodic prophylaxis	0	3	0	1	N/A
Prophylaxis	0	23	1	6	N/A
Immune tolerance induction	0	0	0	1	N/A
NovoSeven	0	0	0	2	N/A

Note: The blood donor population consisted of 215 male blood donors, from which results have been recently published,³² and nine paediatric males, included to attempt to better represent the age range of the haemophilia population. The ethnicity of the blood donors was previously published.³² HIV = human immunodeficiency virus; HCV = hepatitis C virus; On-demand = on-demand factor replacement therapy; Prophylaxis = prophylactic factor replacement therapy.

3.2 |. Measurement of plasma TFPI, FV and PS in haemophilia patients

The average plasma total TFPI, TFPIα, FV and PS for people with haemophilia and 224 male blood donors are presented in Table 2. Among all people with haemophilia, the average plasma total TFPI and plasma TFPIα were 63.7 ng/mL (95% confidence interval (CI): 57.7–69.7 ng/mL) and 12.5 ng/mL (95% CI: 11.7–13.3 ng/mL), respectively. The average plasma FV and plasma PS were 21.4 μg/mL (95% CI: 20.2–22.5 μg/mL) and 19.8 μg/mL (95% CI: 18.3–21.3 μg/mL), respectively. It is noted that the plasma FV concentrations of the blood donor population, as previously published,³² and haemophilia populations, are higher than that reported by others (~10 μg/mL).^41–43 This difference does not affect comparisons of the plasma or platelet FV concentration in the populations investigated in the current study. In unadjusted comparisons of those with haemophilia A or B, those with haemophilia B appeared to have higher total TFPI and PS. However, there was considerable overlap of values for all plasma proteins measured between groups (Figure 1). Therefore, multiple regression models were developed to determine whether haemophilia type (i.e. A or B) or severity (i.e. mild/moderate and severe) was associated with plasma protein concentrations (Table 3). In these analyses, there were no differences in plasma TFPIα, FV or PS between people with haemophilia A or haemophilia B. However, as a cohort, people with haemophilia had approximately 9% lower plasma TFPIα than blood donors (P = .0254). Differences observed in subgroup analyses (i.e. A or B vs donors, mild/moderate or severe vs donors) were not statistically significant. Remarkably, plasma PS was approximately one-third lower in people with haemophilia compared with blood donors (ratio = 0.67, P < .0001). This ratio was consistently and significantly lower by 24%–33% in all subgroup analyses (Table 3).

TABLE 2.

Mean plasma and platelet concentrations of FV, PS and TFPI in people with haemophilia and in blood donors

Variable Measured	Blood Donors	Haemophilia A and Haemophilia B	Haemophilia A	Haemophilia B
Plasma Total TFPI (ng/mL)	61.9 (59.2–64.5; 224)	63.7 (57.7–69.7; 91)	62.0 (56.4–67.6; 73)	70.6 (49.0–92.3; 18)
Plasma TFPIα (ng/mL)	14.5 (14.0–15.1; 224)	12.5 (11.7–13.3; 91)	12.5 (11.6–13.3; 73)	12.9 (10.6–15.2; 18)
Plasma FV (μg/mL)	21.2 (20.4–21.9; 224)	21.4 (20.2–22.5; 91)	21.5 (20.1–22.8; 73)	21.3 (18.3–24.3; 18)
Plasma PS (μg/mL)	29.5 (28.1–30.8; 222)	19.8 (18.3–21.3; 91)	19.4 (17.6–21.1; 73)	21.5 (18.7–24.3; 18)
Platelet TFPI (ng/mg TP)	21.0 (19.3–22.6; 199)	21.4 (19.8–23.0; 90)	21.4 (19.7–23.1; 73)	21.5 (16.7–26.3; 17)
Platelet FV (ng/mg TP)	335.9 (294.9–377.0; 167)	470.1 (369.5–570.7; 81)	449.2 (344.7–553.8; 66)	561.9 (243.3–880.5; 15)
Platelet PS (ng/mg TP)	154.2 (135.7–172.7; 167)	131.1 (106.7–155.4; 81)	136.9 (109.1–164.7; 66)	105.5 (52.7–158.2; 15)

Note: Numbers represent the mean (95% confidence interval; number of samples). The blood donor data are combined from the male population published in Ellery et al (2018) and nine (9) paediatric blood samples. In some instances, there was insufficient sample to measure all analytes, and therefore, the number of samples measured in a single population varies between some analytes.

FIGURE 1.

Dot plots of Plasma Total TFPI (A), TFPIα (B), FV (C) and PS (D) in blood donors and people with haemophilia A or haemophilia B. Each dot represents an individual sample

TABLE 3.

Comparison of Plasma and Platelet FV, PS, TFPIα and Total TFPI between haemophilia types, and between haemophilia populations and blood donors

	A vs B	Mild/Moderate vs Severe	Haemophilia vs Donors	A vs Donors	B vs Donors	Mild/Moderate vs Donors	Severe vs Donors
Plasma Total TFPI (ng/mL)
20 y	0.95	0.93	1.03	1.02	1.07	0.99	1.06
40 y	1.23	0.90	0.92	0.94	0.77	0.87	0.97
60 y	1.59	0.87	0.81	0.87	0.55	0.77	0.88
Plasma TFPIα (ng/mL)	0.98 (0.8277)	1.00 (0.9949)	0.91 (0.0254)	0.91 (0.0586)*	0.93 (0.5119)*	0.92 (0.2153)*	0.91 (0.1086)*
Plasma FV (μg/mL)	1.02 (0.8332)*	1.00 (0.9984)	1.06 (0.1272)	1.06 (0.2453)*	1.05 (0.764)*	1.1 (0.1243)*	1.03 (0.7433)*
Plasma PS (μg/mL)	0.86 (0.1284)	1.07 (0.4474)	0.67 (<0.0001)	0.65 (<0.0001)	0.76 (0.005)	0.67 (<0.0001)	0.67 (<0.0001)
Platelet TFPI (ng/mg protein)	1.03 (0.8429)	0.82 (0.1824)	1.13 (0.084)	1.13 (0.1776)	1.1 (0.7327)	1.00 (0.9997)	1.21 (0.0396)
Platelet FV (ng/mg protein)	0.93 (0.7163)*	1.14 (0.6344)	1.52 (0.0001)*	1.5 (0.001)*	1.62 (0.0335)*	1.64 (0.0016)*	1.44 (0.0095)*
Platelet PS (ng/mg protein)	1.27 (0.3177)	1.04 (0.976)	0.74 (0.0081)	0.77 (0.0682)	0.61 (0.0554)	0.75 (0.1748)	0.73 (0.0384)

Note: The numbers represent the average ratio of the values obtained from the two populations at the top of each column. Numbers in brackets represent the P-value. Numbers in bold are statistically significant. The asterisk represents models where age has a significant effect in both populations to the same degree. Age affected Plasma Total TFPI to a different degree in each of the populations studied, and therefore, ratios at 20, 40 and 60 y of age were calculated and are presented.

3.3 |. Measurement of platelet TFPI, FV and PS in haemophilia patients

The average platelet TFPI, FV and PS also are presented in Table 2. In the haemophilia cohort, the average platelet TFPI was 21.4 ng/mg total protein (95% CI: 19.8–23.0 ng/mg protein). The average FV and PS were 470.1 ng/mg total protein (95% CI: 369.5–570.7 ng/mg protein) and 131.1 ng/mg total protein (95% CI: 106.7–155.4 ng/mg total protein), respectively. While platelet TFPI was nearly identical between haemophilia A and haemophilia B, those with haemophilia B had higher unadjusted platelet FV and lower unadjusted platelet PS (Table 2). Again, there was considerable overlap of values for all platelet proteins measured between groups (Figure 2). Therefore, the multiple regression models were used to determine whether haemophilia type (i.e. A or B) or severity (i.e. mild/moderate and severe) was associated with platelet protein concentrations (Table 3). In these analyses, platelet protein concentrations did not differ between people with haemophilia A and haemophilia B. When compared to blood donors, people with haemophilia had approximately 50% higher platelet FV (ratio = 1.52, P = .0001), which persisted in all subgroup analyses. As a cohort, people with haemophilia had 26% lower platelet PS compared with blood donors (ratio 0.74, P = .0081). This decrease persisted in all subgroup analyses but was only significant in severe haemophilia (ratio = 0.73, P = .0384). Platelet TFPI was not different between people with haemophilia and blood donors. However, in subgroup analyses, those with severe haemophilia had platelet TFPI 21% greater than blood donors (ratio = 1.21, P = .0396).

FIGURE 2.

Dot plots of Platelet TFPI (A), FV (B) and PS (C) in blood donors and people with haemophilia A or haemophilia B. Each dot represents an individual sample

3.4 |. Correlation of total TFPI, TFPIα, FV and PS in plasma and platelets

Correlations between the plasma and platelet protein concentrations were investigated to identify potential interactions within the haemophilia cohort (Table 4). Moderate correlations between platelet PS and platelet FV (r² = .41) and plasma PS and plasma FV were present (r² = .39), while weaker positive correlations between platelet TFPI and platelet PS (r² = .29), plasma total TFPI and platelet TFPI (r² = .28), and plasma TFPIα and plasma FV (r² = .25) were observed. There was no correlation between plasma TFPIα and platelet TFPI (r² = .07). A moderately negative correlation between plasma and platelet PS (r² = −.36) was also present.

TABLE 4.

Pairwise correlations between all analytes investigated

Analyte 1	Analyte 2	Correlation	Lower 95% CI	Upper 95% CI	P-value
Plasma TFPIα	Plasma Total TFPI	0.48	0.30	0.62	<.0001
Platelet PS	Platelet FV	0.41	0.21	0.58	.0001
Plasma PS	Plasma FV	0.39	0.20	0.55	.0001
Platelet PS	Platelet TFPI	0.29	0.07	0.47	.0098
Platelet TFPI	Plasma Total TFPI	0.28	0.08	0.46	.0074
Plasma FV	Plasma TFPIα	0.25	0.05	0.43	.0168
Platelet FV	Plasma TFPIα	0.17	−0.06	0.37	.14
Platelet FV	Platelet TFPI	0.17	−0.15	0.38	.12
Platelet FV	Plasma FV	0.14	−0.08	0.35	.20
Platelet PS	Plasma Total TFPI	0.12	−0.10	0.33	.29
Platelet PS	Plasma TFPIα	0.11	−0.11	0.32	.33
Platelet TFPI	Plasma TFPIα	0.07	−0.14	0.28	.49
Plasma PS	Plasma TFPIα	0.06	−0.15	0.26	.58
Plasma FV	Plasma Total TFPI	0.04	−0.17	0.25	.70
Plasma PS	Plasma Total TFPI	0.03	−0.18	0.23	.81
Plasma PS	Platelet TFPI	0.02	−0.19	0.22	.87
Platelet FV	Plasma Total TFPI	0.00	−0.22	0.22	.99
Platelet FV	Plasma PS	−0.04	−0.26	0.18	.70
Plasma FV	Platelet TFPI	−0.05	−0.26	0.15	.61
Platelet PS	Plasma FV	−0.06	−0.28	0.16	.59
Platelet PS	Plasma PS	−0.36	−0.53	−0.15	.0011

4 |. DISCUSSION

Pharmacological inhibitors of TFPI are currently under development for the treatment of haemophilia (reviewed in⁷). Plasma and platelet TFPI concentrations in 73 people with haemophilia A and 18 with haemophilia B were determined to examine physiological associations with FV and PS concentrations, and to inform decisions for TFPI inhibitor dosing strategies. Importantly, there were no statistically significant differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. Similar to findings by others,³³ when compared to blood donors, people with haemophilia had mildly decreased plasma TFPIα concentration, though this was not evident in subgroup analyses. Furthermore, people with haemophilia had about one-third lower plasma and platelet PS concentration, and 1.5-fold higher platelet FV concentration than blood donors. Together, these findings suggest the presence of a compensatory procoagulant response to haemophilia bleeding.

Comparisons between different groups were made using multiple regression models to account for the potentially confounding age or race/ethnicity-related effects on analyte concentrations that were identified in the blood donor population,³² and to account for the difference in average age between the haemophilia and blood donor populations. There were no differences in plasma total TFPI or TFPIα concentrations between people with haemophilia A or haemophilia B. This finding is in agreement with recent publications by Fosbury⁴⁴ and Chelle and colleagues,⁴⁵ who examined a combined 91 people with haemophilia A and 45 with haemophilia B and also found no differences in the plasma concentrations of TFPI. However, they are in contrast to a study of 30 people with haemophilia A and 21 with haemophilia B by Tardy-Poncet and colleagues,³⁸ who found decreased plasma free-TFPI in people with haemophilia B vs those with haemophilia A. Differences in the distribution of haemophilia types between study populations, the TFPI assays used, or inter-individual variation, might account for this discrepancy. Overall, the preponderance of data now indicates that there is no difference in plasma TFPI concentration between people with haemophilia A and haemophilia B. The findings presented here of similar platelet TFPI, FV and PS, as well as plasma FV and PS, between people with haemophilia A and haemophilia B suggest that anti-TFPI pharmaceuticals will have similar biological effects in either type of haemophilia.

The embryonic lethal phenotype of PS-deficient mice is rescued by concomitant FVIII deficiency,⁴⁶ suggesting that the functions of PS and FVIII are counterbalanced in a manner critical to maintaining haemostasis during embryonic development. Interestingly, compared with blood donors, people with haemophilia exhibited decreased average plasma PS (~67%) and elevated average platelet FV (~150%), irrespective of haemophilia type or severity. As for blood donors, a large degree of inter-individual variation was noted for all analytes measured in the haemophilia population. In addition, while there were no differences in platelet TFPI between all people with haemophilia and blood donors, those with severe haemophilia had increased average platelet TFPI (121%), which represents an anticoagulant difference. Nevertheless, when all data from this study are taken together, the observed differences in people with haemophilia appear to shift the haemostatic balance towards coagulation. It is tempting to speculate that the changes represent a physiological compensatory mechanism to haemophilia bleeding. However, confirmation of these findings in an independent cohort is required.

As in the blood donor population,³² the strongest correlations observed in the haemophilia population were between plasma total TFPI and plasma TFPIα, and between platelet FV and platelet PS. There was also a weak correlation between plasma TFPIα and plasma FV. These findings are consistent with the storage of platelet FV and PS in platelet alpha granules,^47,48 and the reported interactions between plasma TFPIα and FV.^{11,13,14,33,49} As in the donor population,³² a positive correlation was observed between plasma FV and plasma PS. FVa and PS are reported to bind in vitro.^50,51 However, an association between FV and PS in the circulation has not been reported, and therefore, the biological basis of this correlation is unknown. Similarly, weak correlations between platelet TFPI and platelet PS, and platelet TFPI and plasma total TFPI, were observed. Platelet PS is stored within alpha granules,⁴⁸ while platelet TFPI is not,³⁶ and the source of platelet and plasma TFPI differs. Therefore, the biological reasons for these correlations remain to be elucidated, though they may have little relevance given they are weak.

In conclusion, no differences in plasma or platelet TFPI, FV or PS concentrations between individuals with haemophilia A and haemophilia B were present, suggesting that either type of haemophilia should respond similarly to treatment with anti-TFPI agents. In addition, interesting differences in plasma and platelet protein concentrations between blood donors and people with haemophilia were present, suggesting a compensatory procoagulant response to the deficiency of FVIII or FIX.