No Relation between Platelet Activity and Haemophilia B Phenotype

The bleeding tendency in haemophilia is based on the amount of residual clotting factor activity, which categorizes the disease into severe (< 1% factor VIII/IX [FVIII/IX] activity), moderate (1–5%) or mild (6–40%) haemophilia.[1] Although this categorization reflects the bleeding tendency fairly well, there is considerable variability in patients with similar factor levels[2] making it difficult to predict the individual haemostatic phenotype. Approximately 10 to 15% of patients with FVIII levels < 1% have a mild bleeding tendency.[3] This phenomenon is still unexplained; pro-thrombotic factors, FVIII half-life, FVIII genotype and fibrinolytic activity do not correlate with bleeding patterns in individual haemophilia patients.[4] [5] [6] Laboratory prediction of bleeding tendency is of great importance, as it may be used to individualize and optimize treatment.

Platelets may be among the determinants of variability in bleeding phenotype. The importance of platelets in thrombin generation in haemophilia patients was demonstrated before.[7] In haemophilia A patients, we found an up-regulation of platelet activation, which was most prominent in severe haemophilia A patients.[8] When correlating platelet activation to FVIII consumption, there appeared to be an inverse correlation. However, in a subsequent larger study in severe haemophilia A patients only, we could not find differences in platelet behaviour between patients with a severe and a mild bleeding tendency.[9]

So far, there are no studies on platelet activity in haemophilia B patients.

In the current cross-sectional study, we aimed to find parameters predicting individual bleeding phenotype in patients with haemophilia B by (1) comparing platelet function (activation and reactivity) in haemophilia B patients to healthy controls and (2) relating platelet function to the bleeding tendency of the individual patients with severe haemophilia B.

Adult patients from the Van Creveldkliniek were included with all types of haemophilia B. Patients with inhibitors were excluded. The study was approved by the Medical Ethical Committee and written informed consent from consecutive patients and healthy donors was obtained. Annual clotting FIX consumption over the past 6 years was collected in severe haemophilia only. Blood was collected out of the antecubital vein into vacuum citrated tubes through 21G needles, by a specialized research nurse. There was no washout period before blood collection in haemophilia patients.

The concentration of different soluble platelet activation markers including regulated upon activation normal T cell expressed and secreted (RANTES), soluble P-selectin and β-thromboglobulin was measured in platelet-poor plasma as described before.[9] Reactivity of platelets towards several agonists and an inhibitor was assessed with flow cytometry.[8] We performed dose–response analyses for five different platelet agonists. All dose–response curves included a baseline sample, in which no agonist was added. We measured dose–response curves for glycoprotein VI agonist convulxin (CVX; 39.1–9.8–2.4–0.6–0.2–0.04–0.01–0 ng/mL), protease-activated receptor-1 agonist SFLLRN (thrombin-receptor activating peptide [TRAP]; 625–156.3–39.1–9.8–2.4–0.6–0.2–0 µM) with or without 5 ng/mL iloprost, P2Y12 agonist adenosine diphosphate (ADP; 125–31.3–7.8–2.0–0.5–0.1–0.03–0 µM) and thromboxane receptor agonist U46619 (142.7–47.6–15.9–5.3–1.8–0.6–0.2–0 µM).

A statistical analysis was performed with SPSS version 20.0 for Windows (SPSS Inc., Chicago, Illinois, United States). Linear regression analysis was done using dummy coding to compare markers of platelet activation and reactivity between the three groups (healthy controls vs. mild and moderate haemophilia B vs. severe haemophilia B). Platelet activation and reactivity parameters were correlated to clinical phenotype (annual FIX concentrate consumption) in patients with severe haemophilia B using linear regression analysis. A p-value of < 0.05 was considered statistically significant.

A total of 12 healthy controls and 31 patients with haemophilia B were included of whom 19 were severe and 12 were non-severe (6 moderate and 6 mild). Thirteen of the 19 severe haemophilia patients used regular clotting factor prophylaxis. In the year before inclusion, there were no spontaneous bleeds in the 6 mild patients while there was 1 spontaneous epistaxis in the 6 moderate patients. In the patients with severe haemophilia not using regular prophylaxis, a mean of 3.8 spontaneous bleedings occurred (range, 0–11). In the patients with prophylaxis, this was 0.2 (range, 0–3). Mean age was 42.3 years for severe haemophilia B, 51.9 years for non-severe haemophilia and 33.3 years for the controls (p = 0.04).

In an univariate analysis, age was significantly related with all outcome parameters. Therefore, all analyses were adjusted for age.

We found no significant differences in platelet activity and reactivity between haemophilia and controls ([Table 1]). Plasma levels of platelet release products β-thromboglobulin and RANTES, as well as soluble P-selectin, showed no differences between controls and haemophilia B (p-values of > 0.17 for all regression coefficients with 95% confidence intervals). The areas under the curve of the mean fluorescence intensity of the platelets using markers CD62 and αIIbβ3 were calculated for the five agonists (ADP, CVX, TRAP, TRAP iloprost and U44619). There was no significant relation between platelet activity and reactivity between controls and haemophilia. There were no differences in baseline platelet P-selectin expression between healthy controls, patients with mild–moderate haemophilia B or severe haemophilia B.

Table 1 Regression coefficients (β) describing the association between platelet activity and reactivity and having severe or non-severe haemophilia

Agonist	Marker[a]	Group	β (95% CI)[b]	p-Value
N.a.	Beta-TG (IU/mL)	Non-severe	–8.27 (–20.29 to 3.76)	0.17
		Severe	–5.02 (–15.27 to 5.24)	0.33
N.a	RANTES (pg/mL)	Non-severe	–1.43 (–4.08 to 1.22)	0.28
		Severe	–0.73 (–2.99 to 1.53)	0.52
N.a.	sP-selectin (pg/mL)	Non-severe	9.58 (–15.88 to 35.03)	0.45
		Severe	2.85 (–18.87 to 24.56)	0.79
ADP	CD62	Non-severe	–0.37 (–1.37 to 0.64)	0.47
		Severe	0.52 (–0.33 to 1.37)	0.28
	αIIbβ3	Non-severe	–0.97 (–2.68 to 0.74)	0.26
		Severe	0.28 (–1.16 to 1.72)	0.70
CVX	CD62	Non-severe	–0.39 (–0.87 to 0.10)	0.12
		Severe	–0.18 (–0.59 to 0.23)	0.39
	αIIbβ3	Non-severe	–0.26 (–0.75 to 0.23)	0.29
		Severe	–0.15 (–0.56 to 0.26)	0.47
TRAP	CD62	Non-severe	–1.00 (–8.28 to 6.28)	0.78
		Severe	2.07 (–4.06 to 8.21)	0.50
	αIIbβ3	Non-severe	–2.85 (–7.43 to 1.73)	0.22
		Severe	–0.70 (–4.57 to 3.16)	0.71
TRAP iloprost	CD62	Non-severe	–0.33 (–5.92 to 5.27)	0.91
		Severe	1.01 (–3.71 to 5.72)	0.67
	αIIbβ3	Non-severe	–0.05 (–0.21 to 0.11)	0.52
		Severe	–0.06 (–0.19 to 0.07)	0.38
U44619	CD62	Non-severe	–0.43 (–3.39 to 2.54)	0.77
		Severe	–0.11 (–2.61 to 2.39)	0.93
	αIIbβ3	Non-severe	–0.82 (–3.48 to 1.84)	0.54
		Severe	–0.41 (–2.66 to 1.84)	0.71

Abbreviations: ADP, adenosine diphosphate; CVX, convulxin; N.a., not applicable; RANTES, regulated upon activation normal T cell expressed and secreted; TG, thromboglobulin; TRAP, thrombin-receptor activating peptide.

^a Expressed as area under the curve of median fluorescence intensity.

^b Adjusted for age, compared with healthy controls.

In the 19 patients with severe haemophilia, mean FIX consumption was 139.861 IU/year (standard deviation, 52.974). In the patients without prophylaxis it was 109.796 IU/year, whereas in patients with prophylaxis it was 153.738 IU/year. There was no significant relation between any marker of platelet activation or reactivity and FIX consumption (data not shown).

To date, the available evidence on the role of platelets in haemophilia bleeding severity shows conflicting results. In an earlier report in haemophilia A, we documented platelet up-regulation, both seen in basal activation and higher platelet reactivity.[8] This was disputed in an experimental animal model.[10] A subsequent clinical study in severe haemophilia A failed to show any differences in platelet activity between patients with a mild or severe bleeding phenotype.[9] In contrast, others demonstrated through thromboelastography that platelet activity is involved in haemophilia phenotype.[11] This indicates that investigation of platelet function in haemophilia is not straightforward. It depends on many variables including the study population (animals vs. humans), the definition of disease severity (definition of severe or mild bleeding phenotype) and the type of assay used to study the platelets (plasma makers with enzyme-linked immunosorbent assay; flow cytometric assays; real-time perfusion; whole blood aggregation).

Although in this study, we did not find a significant relation between platelet function and bleeding phenotype, this does not exclude that platelets are not involved in regulation of this phenotype. First of all, our studied sample size is small which makes it difficult to find statistically significant differences. Second, the number of spontaneous bleeds in our cohort is very low. Even in the patients with severe haemophilia not using regular prophylaxis, the mean number of spontaneous bleeding episodes was 3.8. However, considering the amount of clotting factor used, it seems that these patients use far more FIX than is expected based on bleeding alone. Probably, the on-demand treatment is used here as non-regular prophylaxis. Third, with our platelet activity test, we analyse the capability to aggregate under exogenous conditions with different agonists. Several important mechanisms involved in primary haemostasis have not been analysed. Among others, these are the interactions between platelets and endothelium through leucine-rich repeat, immunoglobulin and glycoprotein receptors, the effect of shear stress and its related affinity of von Willebrand factor to platelets.

An interesting option to further explore primary haemostasis is the use of real-time perfusion flow chambers. With flow perfusion, several aspects that are lacking in a static function analysis can be dynamically evaluated including platelet adhesion, spreading, aggregate formation and clot retraction. Different flow conditions enables to analyse primary haemostasis in altered settings of shear stress. A standardized procedure to test thrombus formation upon whole-blood perfusion was described,[12] paving the way for future studies in this field. In line with this, a microfluidic assay has indeed shown that haemophilia patients have altered platelet depositions.[13]

In conclusion, with currently used platelet function analysis, we were not able to demonstrate a statistical significant relationship between bleeding phenotype in haemophilia B and platelet function. There is an on-going medical need for global coagulation assays that are able to predict or monitor individual haemostatic properties in haemophilia.

• References

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