Von Willebrand disease combined with coagulation defects in Iran

Abstract

Background

Although Von Willebrand disease (VWD) is the most common inherited bleeding disorder, few cases of VWD combined with coagulation defects have been reported. This study sought to determine the clinical and laboratory features of VWD combined with other coagulation defects and to evaluate the prevalence of this combination in Iran.

Material and methods

A total of 3,120 cases were evaluated to confirm a suspected diagnosis of VWD. Clinical and laboratory phenotypes, including bleeding scores (BS), were also obtained.

Results

A diagnosis of VWD was established for 130 patients. Following their further characterisation, a subgroup of 25 patients with a dual or triple combination of VWD with coagulation defects (FXII, FXI, FIX, FVII, FV, and lupus anticoagulant) was identified. Their laboratory and clinical data were compared with those of healthy controls (n=25) and VWD-only patients (n=25). No differences were observed for VWF-related laboratory measurements between the combined deficient cases and those with VWD only, results being expectedly lower than in healthy controls. The median BS of combined patients was 4, higher than for VWD-only and control groups (median BS 3 and 1; p=0.55 and p<0.001, respectively).

Discussion

The prevalence of combined coagulation defects was 19.2% among all the VWD cases. The co-occurrence of VWD with clotting factor deficiencies may lead to more severe clinical presentations. To ensure adequate treatment, combined defects should be considered in VWD patients presenting with a more severe bleeding phenotype than expected or with a poor response to treatment.

INTRODUCTION

Von Willebrand disease (VWD) is the most common inherited bleeding disorder with an estimated prevalence of about 1% in the general population¹. It is transmitted with autosomal dominant or recessive patterns of inheritance and is characterised by heterogeneous laboratory and clinical phenotypes. It is caused by quantitative (types 1 and 3) or qualitative (type 2) defects of Von Willebrand factor (VWF), a large multimeric plasma glycoprotein that supports platelet adhesion at sites of vascular damage and stabilises coagulation factor VIII (FVIII) in plasma². Deficient or dysfunctional VWF may be due to such different mechanisms as reduced synthesis, accelerated degradation or plasma clearance of this protein or abnormal affinity for its ligands (platelet receptors, collagen or plasma FVIII)³. The most common clinical symptoms in VWD are mucocutaneous bleeding such as epistaxis and menorrhagia, excessive bleeding after invasive procedures or from the gastrointestinal (GI) tract^4,5. The severity of bleeding varies even among members of the same family, although in general it is associated with the type of VWD and the plasma levels of VWF and FVIII⁶. Due to the high prevalence of VWD, it is predicted that patients with other coagulation defects may have this disease concomitantly, although very few well-documented cases of VWD combined with coagulation factor deficiencies have been reported (e.g. FXII and FXI deficiency, haemophilia A)^7–10. No comprehensive study has been carried out to investigate the frequency and clinical manifestations of VWD combined with coagulation defects. We therefore chose to report the clinical and laboratory features of patients with the combination and to evaluate their prevalence in Iran, a country characterised by a high rate of consanguinity.

MATERIALS AND METHODS

Patients

We evaluated 3,120 patients referred from April 2018 to March 2020 to the reference coagulation laboratory of the Iranian Blood Transfusion Organization with the goal of undertaking laboratory evaluation for a suspicion of VWD. In addition, a group of 40 normal subjects with no pre-existing diagnosis of bleeding disorder was included as part of the internal quality control of our reference coagulation laboratory; this group also served to determine the reference ranges for each assay. The diagnosis of VWD was established according to the recent 2021 joint guidelines on the diagnosis of Von Willebrand disease issued by the American Society of Hematology (ASH), the International Society on Thrombosis and Haemostasis (ISTH), the National Hemophilia Foundation (NHF) and the World Federation of Hemophilia (WFH)¹¹. Therefore, patients with a positive personal history of bleeding or a family history of VWD who had plasma VWF levels <50 IU/dL were defined as having VWD.

All samples were collected in 0.105M buffered tri-Na citrate solution and centrifused at room temperature to obtain platelet-poor plasma (PPP), and an aliquot of each sample was stored at −80°C. Most haemostasis tests were performed on the day of blood sampling, although tests for lupus anticoagulant and the VWF collagen binding assay (VWF: CB) were performed on stored plasma not previously thawed. Patients who had received FVIII or VWF concentrates within 10 days from blood sampling and pregnant women were excluded. Written informed consent was obtained in accordance with the Declaration of Helsinki from all the VWD cases and healthy individuals. Ethical approval was also obtained from the High Institute for Research and Education in Transfusion Medicine Ethics Committee in Tehran.

Laboratory assays

All haemostasis testing was performed at the National Hemostasis Reference Laboratory of the Iranian Blood Transfusion Organization, Tehran. First, the panel of tests for the VWD diagnosis, including one-stage FVIII coagulant assay (FVIII: C), Von Willebrand factor antigen (VWF: Ag), and Von Willebrand ristocetin co-factor activity (VWF: Rco), were performed. Samples with a prolonged prothrombin time (PT) and/or activated partial thromboplastin time (aPTT) were further investigated by means of other coagulation factor assays and the lupus anticoagulant test panel. For the measurement of coagulation factor activity (FV, FVII, FVIII, FIX, FXI, FXII), VWF: Ag, VWF: RCo, PT and aPTT Stago commercial kits and the STA Compact Max® analyzer (Stago, Gennevilliers, France) were used. In some patients, if the commercial kit for VWF: Ag was not available, this measurement was obtained by enzyme-linked immunoabsorbent assay (ELISA) using Asserachrom ELISA kits (Stago). In patients with values outside the normal range, all laboratory tests were repeated at least once. A commercial ELISA Von Willebrand factor collagen binding (VWF: CB) with type I (95%) and type III (5%) collagen using Zymutest VWF: CB (Hyphen Biomed, Neuville sur Oise, France) was carried out in samples of patients with VWD combined with coagulation defects in order to obtain an accurate VWD classification. For the diagnosis of lupus anticoagulant, the International Society of Thrombosis and Haemostasis Scientific Standardization Committee (ISTH SSC) recommendations were used¹², so that a diagnosis was first made when a patient had an unexplained prolongation of the aPTT, uncorrected aPTT mixing test (1 : 1 volumes of patient plasma and normal plasma), and positive Russell’s ViperVenom Time (dRVVT) screening. It was further confirmed by means of a dRVVT screen ratio (patient dRVVT screen result/mean of dRVVT screen normal range [NR] and a dRVVT confirm ratio [patient dRVVT confirm/mean of dRVVT confirm NR] >1.2. For the detection of lupus anticoagulant, we used the silica clotting time (HemosIL™, reagent: HemosIL Silica Clotting Time, Instrumentation Laboratory, Werfen, Bedford, MA, USA) and the diluted RVVT (dRVVT, HemosIL™, reagent: HemosIL dRVVT Screen/Confirm, Instrumentation Laboratory).

Bleeding score

The ISTH Bleeding Assessment Tool (ISTH-BAT) was administered to each case to assess the bleeding history. This questionnaire evaluates both the severity and frequency of 14 different bleeding symptoms scored in a range from 0 to 4. According to Elbatarny et al.¹³, the cut-off for a positive or abnormal bleeding score (BS) is ≥4 in adult males, ≥6 in adult females, and ≥3 in children. For statistical analysis, the 2014 IBM SPSS Statistics Premium Grad Pack 22 (IBM, Armonk, New York, USA) and the Mann-Whitney U test were used to compare medians between two independent groups. p-values ≤0.05 were considered statistically significant and data are presented as medians (range).

RESULTS

In total, 130 patients were diagnosed with VWD throughout the whole 2-year study period. Cases were classified as VWD type 1 if the plasma levels of VWF: Ag and VWF: RCo were correspondingly low (VWF: RCo/VWF: Ag ratio >0.7), type 2 if VWF: RCo/VWF: Ag ratio were ≤0.7 and type 3 if VWF: Ag levels were <5 IU/dL. Among the 130 cases, we identified 25 patients from unrelated families with a combination of VWD, diagnosed as above, and other coagulation defects, diagnosed when the corresponding plasma values were below the age-specific normal ranges. The prevalence of VWD combined with coagulation defects was 19.2% (25/130) among all VWD cases. Among the 25 combined cases, 17 were type 1 VWD (68%), 7 type 2 (28%), and 1 type 3 (4%). There were 8 males (32%) and 17 females (68%), with a median age of 17 years (range 2–76). Patients’ characteristics according to laboratory results and bleeding phenotype are summarised in Table I.

Table I.

Clinical and laboratory features at screening of 25 patients with von Willebrand disease (vWD) and combined coagulation defects

Patient n.	Age/sex	Main clinical presentation	FVIII: C (IU/dL)	VWF: Ag (IU/dL)	VWF: RCo (IU/dL)	VWF: CB (IU/dL)	VWF: RCo/ VWF: Ag ratio	vWD type	Blood Group	BS	Coagulation defect (s) (IU/dL)
1	6 years/M	Epistaxis	78	44	28	88	0.64	Type 2	Non-O	3	FXII 34, FV 26
2	5 years/M	Epistaxis, bruising	74	40	30	39	0.75	Type 1	O	3	FXII 42, LA positive
3	76 years/M	Postoperative bleeding, oral cavity bleeding	26	20	15	21	0.75	Type 1	Non-O	8	FXII 34, LA positive
4	27 years/F	Bruising, postoperative bleeding, menorrhagia	64	48	39	87	0.81	Type 1	O	9	FXII 38
5	37 years/F	Menorrhagia	75	42	36	41	0.85	Type 1	Non-O	2	FXII 2
6	3 years/F	Bruising	53	31	41	-	1.3	Type 1	Non-O	2	FXII 38
7	2 years/M	Bruising	70	49	32	42	0.65	Type 2	-	1	FXII 21
8	4 years/F	Bruising	38	32	26	28	0.81	Type 1	O	2	FXII 37
9	28 years/M	Epistaxis	83	51	35	47	0.68	Type 2	O	4	FXII 35, FXI 58
10	27 years/F	Epistaxis, menorrhagia	75	43	35	45	0.81	Type 1	O	7	FXI 2
11	1 years/F	Epistaxis, bruising	3	2	<1	<1	-	Type 3	Non-O	4	FXI 45
12	30 years/F	Menorrhagia epistaxis	38	36	32	-	0.88	Type 1	-	6	FXI 42
13	35 years/F	Menorrhagia, epistaxis, bruising	58	39	34	36	0.87	Type 1	O	10	FXI 28
14	6 years/F	Epistaxis	47	41	37	42	0.91	Type 1	Non-O	3	FIX 36
15	5 years/M	Postoperative bleeding, oral cavity bleeding	78	42	34	28	0.8	Type 1	O	10	FVII 42
16	31 years/F	Bruising, oral cavity bleeding	43	44	36	53	0.72	Type 1	O	7	FVII 11
17	32 years/M	Epistaxis	70	45	32	41	0.71	Type 1	O	4	FVII 51
18	9 years/M	Epistaxis, bruising	56	22	31	33	1.4	Type 1	Non-O	5	FVII 31
19	3 years/M	Epistaxis, bruising	69	64	36	-	0.56	Type 2	Non-O	6	FVII 35
20	6 years/F	Epistaxis, bruising	51	42	35	37	0.83	Type 1	Non-O	4	FVII 47
21	14 years/F	Epistaxis	58	30	16	-	0.53	Type 2	O	3	FVII 42
22	18 years/F	Oral cavity bleeding, menorrhagia	83	36	20	85	0.55	Type 2	Non-O	4	FVII 42
23	3 years/ F	Bruising	79	48	38	45	0.79	Type 1	O	3	FVII 17
24	4 years/F	Epistaxis, oral cavity, GI bleeding	64	61	35	32	0.57	Type 2	O	20	FVII 34, LA positive
25	5 years/M	GI bleeding, postoperative bleeding	54	34	29	36	0.85	Type 1	Non-O	17	LA positive

M: male; F: female; BS: bleeding scores; GI: gastrointestinal; CB: collagen binding assay; LA:

To better characterise the clinical and laboratory features of patients with VWD plus other coagulation defects, we compared them with the same sample size (age-, sex- and VWD type-matched) of patients who had VWD only (n=25) and also a group of healthy controls (n=25). The most common combined factor deficiencies were FVII deficiency (36%), FXII deficiency (24%) and FXI deficiency (16%). Triple defects were identified in 5 patients. Two of them had VWD combined with FXII deficiency and lupus anticoagulant (Patients n. 2 and 3), two had a combination of VWF and FXII defects with FV (Patient n. 1) or FXI deficiency (Patient n. 9), and 1 had VWD combined with FVII deficiency and lupus anticoagulant (Patient n. 24). Combined VWF and FIX deficiencies (Patient n. 14) and combined VWD and lupus anticoagulants (Patient n. 25) were also found.

Laboratory phenotype

Table II shows the comparison of laboratory and clinical results between combined deficient and VWD-only patients. The median VWF: Ag in the combined deficient patient group was similar to that in the VWD-only group (p=0.395) but, as expected, lower than in healthy controls (111 IU/dL, p<0.001). The median levels of VWF functional activities (VWF: RCo and VWF: CB) were lower for combined deficient than for the VWD-only group but differences were not statistically significant (p=0.393 and p=0.322, respectively). As expected, these values were higher in healthy controls, VWF: RCo 85 IU/dL (range 55–135, p<0.001), and VWF: CB 91 IU/dL (range 57–133 p<0.001). The median values of FVIII: C were lower than in healthy controls (114 IU/dL, range 66–158, p<0.001) in both the combined deficient and VWD-only group with no significant differences between the last two groups (p=0.252). The median values of FXII, FXI and FVII in patients who had combined VWF and FXII (Patient n. 9), VWF and FXI (Patient n. 5), and VWF and FVII (Patient n. 10) deficiencies, with or without an additional third defect, were 31 IU/dL (range 2–42), 35 IU/dL (range 2–58) and 34 IU/dL (range 11–51), respectively.

Table II.

Comparison of laboratory and clinical phenotype data in the combined deficient versus Von Willebrand disease (VWD) only groups

	Combined (n: 25)	VWD only (n: 25)	p-value
Sex (M/F)	8/17	8/17	-
Age	17 (1–76)	19 (1–62)	-
FVIII: C (IU/dL)	59 (3–86)	57 (12–88)	0.252
VWF: Ag (IU/dL)	40 (2–64)	39 (11–59)	0.395
VWF: RCo (IU/dL)	31 (1–41)	28 (7–37)	0.393
VWF: CB (IU/dL)	40 (1–88)	32 (7–77)	0.322
VWF: RCo/ VWF: Ag ratio	0.8 (0.53–1.4)	0.68 (0.38–0.88)	0.242
Bleeding score	4 (1–20)	3 (1–8)	0.550

Data are presented as medians ranges. M: male; F: female.

Clinical phenotype

The median BS in the group of patients with combined VWD was 4 (range 1–20). These patients showed a wider range and higher BS than the VWD-only cases (Figure 1), but the difference was not statistically significant (p=0.550) (Table II), whereas, as expected, it was significantly higher than in healthy controls (median 4 vs 1, p<0.001). Considering that patients with FXII deficiency have no bleeding manifestations, a subgroup analysis was carried out within the combined deficiencies between those cases with VWD and FXII-only deficiency (n=5) and those with VWD and other coagulation defects (n=20). There was a statistically significant difference for BS between the two groups (p=0.019). The median of BS for VWD combined with FXII deficiency was 2 (range 1–9) and 4 (range 3–20) for VWD combined with other defects. The most common bleeding manifestations in patients with combined VWD were cutaneous bleeding (72%), menorrhagia (63%), epistaxis (60%), and oral cavity bleeding (24%) (Figure 2). Generally, patients with combined defects had a higher bleeding frequency than VWD-only cases except for patients with minor wounds, whose clinical features were comparable with those of the VWD-only cases. Three patients (12%) with combined defects and 1 in the VWD-only group (4%) experienced GI bleeding. However, other severe bleeding manifestations (such as central nervous system bleeding, haemarthrosis and haematomas) were not recorded in the combined deficient nor in VWD-only patients. Using the ISTH-BAT criteria in patients with combined VWD, 79% (13/17) of females and 87% (7/8) of males had a clinically significant bleeding score according to the ISTH-BAT. No thrombotic events were observed in patients deficient in FXII or FVII or with lupus anticoagulant.

Figure 1.

Box plot of bleeding scores (BS) in Von Willebrand disease (VWD) combined with coagulation defects, VWD only, and healthy controls

Solid line indicates median.

Figure 2.

Reported frequency of bleeding manifestations with an International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) subscore ≥1 in healthy controls (open bars), combined deficient (solid bars), and Von Willebrand Disease (VWD)-only group (grey bars)

DISCUSSION

In the frame of an evaluation of 3,120 Iranian cases referred for a suspected diagnosis of VWD, 130 VWD cases were identified; among these there was a subgroup of 25 patients with the combination of coagulation defects with various VWD types. To our knowledge, this is the largest reported series of patients with VWD combined with coagulation defects; indeed, only a few isolated cases have so far been reported^7–10,14,15. O’Brien et al. reported 6 patients with a combination of factor deficiencies with probable or definite type 1 VWD and estimated a 2.6% prevalence for the combination (6/227)⁷. A likely reason for the higher prevalence found in our study may be due to the frequency of consanguineous marriages in Iran, as shown by the fact that 36% of the patients’ parents were first or second-degree cousins.

There was a wide range of combinations of VWF and coagulation defects, including dual or triple defects (Table I). The most common combination was with FVII deficiency followed by FXII and FXI deficiencies. Comparable results were obtained in other surveys carried out in Germany and France, showing that VWD combined with contact phase defects (FXII or FXI) is relatively common^8,9.

Thus, we suggest that a prolonged PT in patients with FVII deficiency may mask the concomitance presence of VWD because physicians tend to overlook secondary or tertiary bleeding causes. On the other hand, in spite of the fact that patients with VWD may have a prolonged aPTT owing to their secondary FVIII defect, the possibility that other factor deficiencies such as FXII, FXI and FIX may affect the aPTT results should be considered.

An accurate diagnosis of combined deficiencies is warranted because the strategy for the management of bleeding episodes may differ from that of a single defect, especially during challenging procedures such as surgery and during pregnancy. Moreover, the bleeding tendency in patients with the co-occurrence of VWD with coagulation factor deficiencies might be more severe than expected, as emphasised by Tavori et al.¹⁵ who showed that in a group of 28 patients with comparably mild FXI deficiency, the 14 patients who experienced bleeding had lower VWF levels (VWF: Ag, VWF: RCo) than the 14 non-bleeders. Therefore, in the clinical setting, when an unexpectedly severe bleeding phenotype or unresponsiveness to standard therapies are observed in patients known to have a single bleeding disorder, the possible presence of combined deficiencies should be considered.

Available studies recommend considering an abnormal BS to be ≥4 in adult males, ≥6 in adult females, and ≥3 in children^13,16. When we applied the ISTH-BAT the vast majority of our patients (79% of females and 87% of males) had an abnormal BS. Considering that most of them had mildly reduced VWF levels, their clinical presentations as bleeders is likely to be the result of co-inheritance of other coagulation defects. In cases with combined defect, the overall median BS was 4, higher than in the VWD-only and healthy control groups (medians: 3 and 1, respectively). Since more than half of these patients were relatively young (56% were <10 years old) and thus unlikely to have already experienced challenges of haemostasis (such as pregnancy, tooth extraction, surgery or menorrhagia), the overall median BS was not particularly high. However, cutaneous bleeding, epistaxis, menorrhagia and oral cavity bleeding were commonly reported (Figure 2) and all the bleeding symptoms except minor wounds were more frequent in the combined defect group than in the VWD-only group. Kadir et al. showed that women with menorrhagia should be assessed not only for VWD but also for VWD combined with FXI deficiency¹⁷; 17% of 150 women with menorrhagia had an inherited bleeding disorder, mainly VWD, FXI deficiency or their combination¹⁷.

Factor XII deficiency is a rare genetic blood disorder that, despite presenting with prolonged aPTT results, is not associated with a bleeding tendency⁸. In this study, 9 cases had VWD combined with FXII deficiency and 4 of these had a third coagulation defect. Cases with combined VWD and FXII-only deficiency (n=5) showed a statistically significantly lower BS than those with VWD combined with other coagulation defects (n=20) (median 2 vs 4, p=0.019).

Patients with lupus anticoagulant may be symptom free but are generally more prone to thrombosis than bleeding. In this study, no thrombosis was reported and, in contrast, all the 4 patients with combined factor deficiencies and lupus anticoagulant had a high BS (with values of 20, 17, 8, and 3) and were bleeders; three of them were children (>5 years old) and the other was 76-years old. Becton and Stine¹⁸ described 6 children with lupus anticoagulant with bleeding symptoms but no thrombosis and suggested that their haemorrhagic presentation, rather than hypercoagulability, might be due to the transient presence of the lupus anticoagulant. In agreement, Male et al.¹⁹ evaluated the spectrum of clinical manifestations associated with the lupus anticoagulant in 95 children and found that bleeding rather than thrombotic events were more frequent (in 10 and 5%, respectively)¹⁹.

CONCLUSIONS

In conclusion, the combined presence of VWD with coagulation defects is not so rare, at least in Iran, and is likely to be underdiagnosed. The co-occurrence of VWD and coagulation factor deficiencies may lead to clinically significant bleeding and therefore to the potential risk of adopting inappropriate treatment strategies. Thus, we suggest that adult and pediatric haematologists should consider the combination of factor deficiency in patients with VWD who present with a severity of bleeding greater than expected or in those who are unresponsive to the standard treatment for VWD.