Platelet Von Willebrand Factor Research Articles

ADAMTS-13 and Von Willebrand factor in relation to platelet response during plasma exchange in thrombotic thrombocytopenic purpura: a clue for disease mechanism?

ADAMTS-13 and Von Willebrand factor in relation to platelet response during plasma exchange in thrombotic thrombocytopenic purpura: a clue for disease mechanism?

Platelet von Willebrand factor determination does not improve the diagnosis of patients with suspected Type 1 von Willebrand disease

Establishing a laboratory diagnosis of a bleeding disorder can be challenging for some patients who present with mucocutaneous bleeding symptoms. A common clinical scenario is an older patient with a prior diagnosis of von Willebrand disease (VWD) or a family history of VWD, who now has von Willebrand factor (VWF) values repeatedly within the normal range. Plasma VWF antigen levels have been shown to increase with age. Whether platelet VWF increases with age is unknown. We hypothesized that platelet VWF does not increase with age and low platelet VWF levels, despite normal plasma levels, could be a reason for continued bleeding symptoms in some patients. Therefore, we compared the platelet and plasma VWF antigen and activity as well as the platelet function analyzer (PFA)-100 closure times in 35 patients with a history of mucocutaneous bleeding symptoms and consistently normal levels of VWF antigen and activity, despite a prior history of a VWD diagnosis and/or a positive family history of VWD. Overall in our patients (bleeders), the platelet VWF values correlated with the plasma values and only three patients had reduced platelet VWF. In the bleeding group, the PFA-100 results showed an inverse correlation with plasma and platelet values, which was stronger for the plasma values. Therefore, platelet VWF determination was not helpful in the diagnosis of suspected mild type 1 VWD.

Morphological Changes of Platelets from Patients with Von Willebrand Disease Types 2B and 3 Suggest That Von Willebrand Factor Intervenes in Platelet Formation from Megakaryocytes.

The interaction between von Willebrand factor (VWF) and platelets plays a key role in hemostasis. VWF gene abnormalities result in the most common inherited bleeding disorder, von Willebrand disease (VWD). VWD2B, characterized by gain-of-function binding of VWF to platelet glycoprotein (GP)Ib, is due to mutations located within a portion of exon 28 of the VWF gene, coding for the VWFA1 domain. We have previously shown in a family with a R1308P mutation that the enhanced interaction between VWF and GPIb can also occur in the bone marrow leading to a premature release of platelets from megakaryocytes (MKs) with circulating large platelets and agglutinates (Nurden et al, Blood 2006). We have now widened our study to see if these changes are common in VWD2B. As a control, we also included platelets from patients lacking VWF. In total, we have analyzed the platelets from 9 VWD2B patients with the following mutations: P1266L, R1306W, R1308C, I1309V, V1316M, R1341Q, R1341W; platelets from 2 VWD3 patients with a large deletion of the VWF gene were also included. We have used electron microscopy (EM) and immunofluorescence (IF) to evaluate platelet morphological characteristics; included in the morphometric studies was the measure of platelet size. We have also analyzed platelet VWF content and performed plasma/platelet VWF multimeric analyses. PMCA4B belonging to the SERCA proteins (a substrate of caspase) and GRP78 a stress protein were evaluated as markers of abnormal megakaryocytopoiesis. At the time of blood withdrawal, 4 patients were thrombocytopenic; nevertheless, for all patients with VWD2B a pool of giant platelets was found. Agglutinates were often but not always present. Globally, the percentage of platelets with the longest diameter (LD) > than 3μm was 58 ±16% (controls 24 ± 13%) and the shortest diameter (SD) > 2μm was 33% ± 13 (controls 4 ± 0.6%). Thus platelets were enlarged without being round. The largest platelets were found for the patient with the V1316M mutation. The content of alpha granules appeared normal, as was the quantity of internal membrane pools. To our surprise, giant platelets were also present for the two VWD3 patients; 66 and 57% of the platelets have a LD > 3μm and 21 and 19% a SD > than 2μm. The EM evaluation for both type 3 patients suggested that some giant platelets resembled large fragments prematurely detached from MKs. Examining further by IF and EM coupled with immunogold labelling with a mixture of anti-GPIIb-IIIa and GPIb-IX MoAbs revealed occasional circulating MKs. Nuclei surrounded by cytoplasmic remnants were also seen. Analysis of PMCA4B and GRP 78, showed that their levels were increased for VWD2B patients with the R1341Q mutation but borderline for the patient with the R1308C mutation (contrasting with increased levels for R1308P) showing that premature apoptosis was not a constant feature. These results were normal for the VWD3 patients. Our results confirm that megakaryocytopoiesis can be affected by both an abnormal gain-of-function (VWD2B) and completely deficient (VWD3) VWF. However, the consequences of up-regulated VWF and VWF deficiency may be different.

Intensive plasma exchange increases a disintegrin and metalloprotease with thrombospondin motifs-13 activity and reverses organ dysfunction in children with thrombocytopenia-associated multiple organ failure*

Thrombocytopenia-associated multiple organ failure (TAMOF) is a poorly understood syndrome in critically ill children. A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-13), formerly known as von Willebrand factor (VWF) cleaving protease, is decreased in adults with VWF-mediated thrombotic microangiopathy, and intensive plasma exchange (PEx) both replenishes ADAMTS-13 and improves outcome in these patients. To determine whether: 1) critically ill children with TAMOF syndrome have decreased ADAMTS-13 activity, 2) ADAMTS-13 activity correlates with platelet counts and VWF antigen, 3) the autopsies from patients who died with reduced ADAMTS-13 activity have VWF-rich microthrombi, and 4) intensive PEx will restore ADAMTS-13 activity and facilitate organ failure resolution. First study: observational. Second study: randomized control trial. Single center university pediatric intensive care unit. First study: thirty-seven consecutive children (17 males and 20 females; ages ranging from 9 days to 23 years) identified with > or = 2 organs dysfunction were enrolled. Seventy-six percent of these children had thrombocytopenia (platelet counts < 100,000/mm3). Five additional critically ill children without MOF were also enrolled. In the second study, children with severe TAMOF (platelet counts < 100,000/mm3 and > 3 organ failure) were randomized to PEx or standard therapy. Primary physicians and parents agreed to enrollment in 10 of the 20 eligible patients with ages ranging from 1 year to 18 years. Five patients received PEx and 5 patients received standard therapy. First study: children with TAMOF (n = 28) had decreased ADAMTS-13 activity, but similar plasminogen activator inhibitor-1 activity and prothrombin time compared to children with MOF without thrombocytopenia (n = 9, p < 0.05). All non-survivors (n = 7) had TAMOF, reduced ADAMTS-13 activity, and VWF-rich microvascular thromboses at autopsy. In the second study, PEx (n = 5, median 12 days, 4-28 days) restored ADAMTS-13 activity and organ function, compared to standard therapy (n = 5, p < 0.05). Children with TAMOF syndrome can have VWF-mediated thrombotic microangiopathy. Similar to adult experience, PEx can replenish ADAMTS-13 activity and reverse organ failure.

Novel point mutation in a leucine-rich repeat of the GPIb chain of the platelet von Willebrand factor receptor, GPIb/IX/V, resulting in an inherited dominant form of Bernard-Soulier syndrome affecting two unrelated families: the N41H variant

In Italy, a significant proportion of patients with autosomal dominant inheritance of macrothrombocytopenia have been recognized as having heterozygous Bernard-Soulier syndrome carrying the Bolzano-type defect. This condition prompted a systematic review of our out-patients with chronic isolated macrothrombocytopenia. We recognized that the affected members of two unrelated families represented a new variant of heterozygous Bernard-Soulier Syndrome with autosomal dominant inheritance. Sequencing analysis of the GPIbalpha gene revealed a novel heterozygous mutation, A169C, resulting in an N41H substitution in the protein. This aminoacid belongs to the first leucine-rich repeat of the chain. The molecular modeling suggests that the replacement of the N41 with a histidine (N41H) drastically disturbs the structure of the first portion of GPIbalpha N-terminal, directly involved in von Willebrand factor binding. As a consequence, platelet aggregation to 1.2 mg/mL of ristocetin is slightly impaired and flow cytometry reveals a reduced binding of monoclonals directed against N-terminal epitopes of the GPIbalpha.

A phase II prospective open‐label escalating dose trial of recombinant interleukin‐11 in mild von Willebrand disease

von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion and is decreased in von Willebrand disease (VWD). 1-8 deamino-d-arginine vasopressin (DDAVP), the most common treatment for VWD, is limited by tachyphylaxis and inconvenience, and in 20% of the patients, unresponsiveness. Recombinant human interleukin-11 (rhIL-11), a gp-130 signalling cytokine with haematopoietic and anti-inflammatory activity, increases VWF antigen and its activity in heterozygous VWF(+/-) mice and dogs. To determine the biological efficacy and safety of rhIL-11 in non-bleeding human subjects with mild VWD, we conducted a phase II prospective open-label trial of rhIL-11 at 10, 25 and 50 mug kg(-1) subcutaneously (s.c.), given daily for 7 days in nine subjects with mild VWD. VWF and factor VIII (FVIII) levels increased gradually and progressively after s.c. rhIL-11, which was sustained through 7 days of dosing to 1.5- to 3-fold over baseline. Following intravenous DDAVP, 0.3 mug kg(-1), on day 7 there was a further boost in VWF and FVIII levels, suggesting that the mechanism of rhIL-11 differs from that of DDAVP. Platelet VWF mRNA expression measured by quantitative PCR increased from two- to eightfold over baseline, suggesting that the mechanism of rhIL-11 effect may be upregulation of VWF mRNA. VWF and FVIII levels returned to baseline by day 14. rhIL-11 was well tolerated with less than grade-1 hypertension, hypokalaemia and fluid retention. Recombinant IL-11 increases VWF levels in humans with mild VWD, justifying future clinical trials to determine its potential in preventing or reducing bleeding in this patient population.

Acute effects of hyperglycaemia on plasma concentration of soluble P-selectin and von Willebrand Factor in healthy volunteers —a prospective randomised double blind controlled study

This study set out to clarify if isolated hyperglycaemia may directly induce acute endothelial and/or platelet activation as diabetes mellitus is a major risk factor for cardio- and cerebrovascular diseases with thromboembolic complications. 12 healthy volunteers were investigated in a prospective randomised, double blind, three-way cross-over trial with a wash-out period of 21 days. Normoglycemic (4.72 mmol/L), moderate (11.1 mmol/L) or high grade (16.7 mmol/L) glucose clamps were maintained for 6 hours by infusion of glucose, insulin and somatostatin. Volunteers were observed for 24 hours. An increase in soluble (s)P-selectin and von Willebrand Factor (VWF) concentrations, of 26 ± 15% and 21 ± 7%, respectively was observed 24 hours after euglycaemic treatment, of 20 ± 7% and 19 ± 5%, respectively after moderate hyperglycaemia and of 22 ± 13% and 18 ± 7%, respectively after high grade hyperglycaemia at 24 hours (p < 0.6 and p < 0.004 in all periods and p = 0.2–0.9 between periods). In conclusion, acute hyperglycaemia for 6 hours does not increase the platelet and endothelial activation markers sP-selectin and VWF in healthy volunteers.

The role of Akt in the signaling pathway of the glycoprotein Ib-IX–induced platelet activation

The platelet von Willebrand factor (vWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. We show that GPIb-IX-induced platelet aggregation and stable adhesion under flow were impaired in mouse platelets deficient in PI3K effectors, Akt1 and Akt2, and in human platelets treated with an Akt inhibitor, SH-6. Akt1 and Akt2 play important roles in early GPIb-IX signaling independent of Syk, adenosine diphosphate (ADP), or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of Akt1 or Akt2 diminished platelet spreading on vWF but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both vWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1- or Akt2-deficiency diminished vWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by exogenous cGMP. Thus, Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway.

Diagnosis and treatment of von Willebrand disease: new perspectives and nuances

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

The Effect of von Willebrand Factor (VWF) on Site-Specific Factor VIII (FVIII) Expression in Hemophilia A Mice.

von Willebrand factor (VWF) is a carrier protein for factor VIII (FVIII) and protects plasma FVIII from protease degradation. Our laboratory has had a longstanding interest in the association of FVIII with VWF both in vitro and in vivo. Our in vitro studies have demonstrated that FVIII stores together with VWF in both endothelial cells and megakaryocytes if FVIII is made in these cells. Furthermore, we demonstrated that FVIII and VWF are both releasable by agonist stimulation. To investigate the association of VWF and FVIII in vivo, we generated two lines of transgenic mice that express FVIII either in endothelial cells or in platelets using either the endothelial cell-specific Tie2 promoter or the platelet-specific αIIb promoter, respectively. When the platelet-specific FVIII (2bF8) transgene is bred into the FVIIInull mouse, FVIII can only be detected in platelets, with a level of 0.76 ± 0.27 mU/108 platelets in heterozygous and 1.53 ± 0.14 mU/108 platelets in homozygous 2bF8 mice. When the endothelial cell-specific FVIII (Tie2F8) transgene is bred into the FVIIInull mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in heterozygous mice (0.56 ± 0.16 U/ml). Both 2bF8trans and Tie2F8trans phenotypes effectively abrogate the bleeding diathesis in hemophilic mice. When 2bF8 transgene was bred into a FVIII and VWF double knockout background, the level of platelet-FVIII significantly decreased, but this platelet-derived FVIII was still stored in a-granules and still maintained clinical efficacy. In contrast, when the Tie2F8 transgene was bred into the double knockout background, plasma FVIII dropped to undetectable levels. This is in contrast to the situation in VWFnull mice in which normal endogenous murine FVIII is synthesized with about 10% of normal FVIII activity persisting in plasma. This could be due to a difference in survival between human FVIII and murine FVIII. All Tie2F8trans/FVIIInullVWFnull mice (n=15) survived tail clipping even though there is no FVIII:C detected in the plasma. To investigate the effect of murine VWF on the levels of plasma FVIII, plasma from FVIIInull mice was infused into Tie2F8trans/FVIIInullVWFnull mice to restore VWF levels to 25% of normal. As expected, the endothelial cell-derived plasma FVIII was stabilized by the infused VWF and was detected within 1 hour after infusion, with a peak (25% level) at 4 hours. The level of plasma FVIII at 24 hours was still about 20% of normal while the level of remaining VWF was only 5% of normal. These results demonstrate that VWF is important for site-specific FVIII expression. Co-expression with VWF in platelets is important for optimal platelet-specific FVIII expression and endothelial cell-derived plasma FVIII is VWF-dependent.

The Roles of Akt1 and Akt2 in GPIb-IX-Mediated Platelet Activation Signaling.

The platelet von Willebrand factor (VWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. To understand the downstream signaling pathway of GPIb-IX signaling, we investigated the roles of PI3K effector kinases, Akt1 and Akt2, in VWF/GPIb-IX-induced platelet activation. VWF/GPIb-IX-induced platelet aggregation was impaired in Akt1- or Akt2-knockout mouse platelets and in human and mouse platelets treated with an Akt inhibitor, SH-6. GPIb-IX-mediated platelet stable adhesion to VWF under shear stress was also inhibited in mouse platelets deficient in Akt1 or Akt2, and in human platelets treated with SH-6. Interestingly, while deficiency of Akt1 or Akt2 caused nearly complete inhibition of stable platelet adhesion to VWF under shear stress, stable platelet adhesion was only partially reduced in platelets treated with both P2Y1 and P2Y12 ADP receptor antagonists, A3P5P and 2MeSAMP or thromboxane A2 pathway inhibitor, aspirin or Syk inhibitor, piceatannol. Therefore, Akt1 and Akt2 are important in early GPIb-IX signaling independent of Syk, ADP or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of either Akt1 or Akt2 diminished platelet spreading on VWF, but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both VWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1 or Akt2 deficiency diminished VWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by low concentration of exogenous cGMP, indicating that Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway. In conclusion, both Akt1 and Akt2 mediate VWF/GPIb-IX-induced signaling pathway leading to platelet activation and the consequent stable platelet adhesion, spreading and aggregation.

Abnormal Large Platelets Are Associated Not Only with von Willebrand Disease (VWD) Type 2B (Including Type 2B/1 New York) but Also with Severe Type 3: A Potential Role for von Willebrand Factor (VWF) in Megakaryocytopoiesis.

Background: VWD type 2B results from mutations in exon 28 of the VWF gene. Gain of function of this adhesive protein results in an increased affinity for the platelet glycoprotein (GP) Ib-IX-V complex. Recently we reported that impaired megakaryocytopoiesis results from an abnormal interaction between GPIb with newly synthesized VWF in megakaryocytes of a family with the R1308P mutation (Blood 2006; 108:2587–95).Aim of the Study: to further examine the potential consequences of VWF abnormalities on platelet production we have studied a series of patients with different types of VWD.Patients, Methods: 13 VWD patients were enrolled in the study after informed consent. Diagnoses of VWD were performed according to the criteria of the ISTH-SSC-SC. Included were 8 VWD 2B patients from 6 families with the following mutations: R1306W (n=1), R1308C (n=1), I1309V (n=1), V1326M (n=2), R1341Q (n=2) and P1266L (n=1, 2B/1NY). Also studied were 5 additional VWD cases characterized by low/absent VWF in their platelets: VWD 2M (n=1, D1277-E78delInsL), VWD 3 without inhibitors against VWF (n=2, 276delT/257delA and 6182delT/6182delT) and VWD with isoantibodies against VWF (n=2, large deletions of the VWF gene). The platelet count was decreased at the time of examination for 6/8 VWD 2B patients and normal for 2/8 (n=1 V1316M and n=1 P1266L). Platelet counts were normal in the remaining 5 patients with VWD types 3, and 2M. Electron microscopy (EM) of platelets and immunolocalization of VWF were performed.Results: EM showed the presence of an increased population of giant platelets (15 to 40% versus <10% for controls) in all VWD 2B. Characteristics of these platelets were the presence of large vacuoles often filled with material and the presence of numerous membrane complexes. Additional abnormalities were observed in the patient with 2B/1NY; alpha-granule morphology was different with a population of enlarged granules, sometimes giant. There was also a heterogeneneous distribution with some platelets almost devoid of alpha-granules. Immunogold staining for all type 2B patients showed that VWF was present not only inside the granules but also in the surface-connected canalicular system. For 3/8 patients with VWD 2B, cleaved caspase was present in the platelets indicating abnormal caspase activity at least for R1341Q and V1316M. In VWD 2M (mutation D1277-E78delInsL) characterized by low platelet VWF content as well as in the VWD 3 (n=2) with a premature stop codon, no significant modification of platelet morphology was found. Some residual VWF was also seen in the alpha-granules of these 2 VWD 3 patients. In contrast, a significant number of enlarged platelets with numerous vacuoles were found in the 2 VWD 3 with large deletions and isoantibodies directed against VWF. Immunogold labelling for platelet VWF was completely negative for these two patients.Conclusions: Patients with VWD types 2B and 3 (undetectable VWF) show platelet production defects of varying severity, suggesting a major role of VWF in the fine regulation of megakaryocytopoiesis. Up-regulation or loss of the interaction between VWF and GPIb may lead to a variable proportion of giant platelets with or without thrombocytopenia.

Common VWF Haplotypes in Normal African-Americans and Caucasians Recruited into the ZPMCB-VWD and Their Impact on VWF Laboratory Testing.

Von Willebrand disease (VWD) is a common bleeding disorder that has been reported to affect up to 1% of the population. Diagnostic testing for VWD relies on specific tests of von Willebrand factor (VWF) that include VWF antigen (Ag) and VWF ristocetin cofactor activity (RCo). Variability in these tests, especially in the RCo, has the potential to affect diagnosis of VWD. Clinically, the RCo/Ag ratio is used to identify patients with type 2M VWD, of which 35% of our local type 2M index cases were of African-American descent. As part of the ZPMCB-VWD, a large study of both healthy controls and patients with VWD, we evaluated VWF Ag, RCo, multimers, EU bleeding score, and VWF gene sequencing to look for mutations and/or common polymorphisms (SNPs) that might contribute to RCo measurement. Healthy controls completed a computerized version of the EU bleeding score and provided blood for clinical VWF testing. Since platelet VWF binding primarily involves the A1 domain of VWF, exon 28 gene sequencing was analyzed and common SNPs were identified, particularly in African-Americans (AA) with altered RCo/Ag ratios. Statistical comparisons were performed using t-tests. For the AA control group, the presence of specific exon 28 SNPs, including I1380V, N1435S, and D1472H, correlated with a low RCo/Ag. In controls, the 3 SNPs occurred together in 22% of AA and 1.5% of Caucasians. For AA controls with all 3 SNPs, the mean Ag was 155, RCo 115, and RCo/Ag 0.77, while for AA without the 3 SNPs, the mean Ag was 129, RCo 129, and RCo/Ag 1.01. The difference in RCo/Ag ratio was significant (p<0.001). In comparison, the Caucasian controls without the 3 SNPs had a mean Ag of 108, RCo of 115, and RCo/Ag of 1.08. When only D1472H was considered, a significant difference in RCo/Ag ratio was noted for both African-American and Caucasian controls with D1472H (present in 63% of AA and 24% of Caucasian controls). For AA controls with D1472H, the mean RCo/Ag ratio was 0.82 (range 0.42–1.16) compared to 1.01 for those without the SNP (p<0.001). The Caucasian controls with D1472H had a mean ratio of 0.87 (range 0.57–1.17) compared to 1.08 for those without the SNP (p<0.001). EU Bleeding Score was not significantly affected by either race or SNP status. All Caucasian controls with D1472H were heterozygous, while 14% of the African-American controls were homozygous. The mean RCo/Ag for the homozygous controls was 0.71, compared to 0.86 for the AA heterozygous controls (p<0.025). In order to determine if the exon 28 SNPs intrinsically altered the measurement of VWF, the 3 SNPs were engineered into recombinant VWF and expressed in HEK293T cells. VWF Ag and RCo were performed on the purified expressed VWF. The RCo/Ag ratios for the recombinant expressed VWF were decreased for 1380V and 1472H, while the construct containing all 3 SNPs showed an even lower ratio (59% of wild-type RCo/Ag ratio). These data suggest that specific exon 28 SNPs may contribute to low RCo/Ag ratios, especially in the African-American population. Since the RCo assay involves ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of “VWF activity” by this assay and might not reflect true hemorrhagic risk.

Fractionation of snake venom metalloproteinases by metal ion affinity: A purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni 2+-agarose

Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni 2+-agarose, and was eluted by ∼10 mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ib α, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIb α extracellular fragment (glycocalicin), and inhibition of 125I-VWF binding to GPIb α on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni 2+-agarose. Samples of flow-through, wash, and imidazole-eluted (0–30 mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni 2+-agarose to fractionate snake venom metalloproteinases.

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

The presence of active von Willebrand factor under various pathological conditions

To highlight mechanisms that regulate the balance between latent and active von Willebrand factor (VWF), and describe pathological conditions leading to increased levels of active VWF. Levels of circulating active VWF are increased in von Willebrand disease type 2B, HELLP syndrome, malaria and antiphospholipid syndrome. Freshly secreted VWF consists of ultra-large multimers that interact spontaneously with platelets at the endothelial cell surface. Proteolysis of ultra-large VWF by a member of the disintegrin and metalloprotease with thrombospondin motif family (ADAMTS13) reduces both multimeric size and accessibility of platelet-adhesion sites. The resulting VWF molecules circulate as inactive multimers, which regain their platelet-adhesion capacity upon binding to the subendothelial matrix, in particular under conditions of high shear. Unfortunately, mechanisms responsible for suppression of circulating plasma levels of active VWF are hampered in a number of pathological conditions, leading to VWF-platelet aggregates associated with thrombotic complications or thrombocytopenia. A recently developed assay allowed us to monitor the presence of circulating active VWF and we found that several diseases are characterized by increased levels. Further analysis provided insight into mechanisms contributing to the presence of active VWF, which revealed that beta2-glycoprotein I may act as a natural regulator of VWF-platelet interactions.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

A role for glycosphingolipid-enriched microdomains in platelet glycoprotein Ib-mediated platelet activation.

Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. Platelets were stimulated by VWF/botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-beta-cyclodextrin (MbetaCD). GPIb was partially translocated to GEMs upon VWF/botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCgamma2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MbetaCD strongly inhibited tyrosine phosphorylation of Syk and PLCgamma2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MbetaCD. The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.

Intravenous DDAVP and Factor VIII-von Willebrand Factor Concentrate for the Treatment and Prophylaxis of Bleedings in Patients With von Willebrand Disease Type 1, 2 and 3

The current standard set of von Willebrand factor (VWF) parameters used to differentiate type 1 from type 2 VWD include bleeding times (BTs), factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetine cofactor activity (VWF:RCo), VWF collagen binding activity (VWF:CB), ristocetine induced platelet aggregation (RIPA), and analysis of VWF multimers in low and high resolution agarose gels and the response to DDAVP. The BTs and RIPA are normal in asymptomatic carriers of a mutant VWF allele, in dominant type 1, and in recessive type 2N VWD, and this category has a normal response of VWF parameters to DDAVP. The response of FVIII:C is compromised in type 2N VWD. The BTs and RIPA are usually normal in type Vicenza and mild type 2A VWD, and these two VWD variants show a transiently good response of BT and VWF parameters followed by short in vivo half life times of VWF parameters. The BTS are strongly prolonged and RIPA typically absent in recessive severe type 1 and 3 VWD, in dominant type 2A and in recessive type 2C (very likely also 2D) VWD and consequently associated with low or absent platelet VWF, and no or poor response of VWF parameters to DDAVP. The BTs are prolonged and RIPA increased in dominant type 2B VWD, that is featured by normal platelet VWF and a poor response of BT and functional VWF to DDAVP. The BTs are prolonged and RIPA decreased in dominant type 2A and 2U, that all have low VWF platelet, very low VWF:RCo values as compared to VWF:Ag, and a poor response of functional VWF to DDAVP. VWD type 2M is featured by the presence of all VWF multimers in a low resolution agarose gel, normal or slightly prolonged BT, decreased RIPA, a poor response of VWF:RCo and a good response of FVIII and VWF:CB to DDAVP and therefore clearly in between dominant type 1 and 2U. The existing recommendations for prophylaxis and treatment of bleedings in type 2 VWD patients with FVIII/VWF concentrates are mainly derived from pharmocokinetic studies in type 3 VWD patients. FVIII/VWF concentrates should be characterised by labelling with FVIII:C, VWF:RCo, VWF:CB and VWF multimeric pattern to determine their safety and efficacy in prospective management studies. As the bleeding tendency is moderate in type 2 and severe in type 3 VWD and the FVIII:C levels are near normal in type 2 and very low in type 3 VWD patients. Proper recommendations of FVIII/VWF concentrates using VWF:RCo unit dosing for the prophylaxis and treatment of bleeding episodes are proposed and has to be stratified for the severity of bleeding, the type of surgery either minor or major and for type 2 and type 3 VWD as well.

Performance and utility of a cost-effective collagen-binding assay for the laboratory diagnosis of Von Willebrand disease

The collagen-binding assay, a functional assay of Von Willebrand factor (VWF), discriminates the subtypes of Von Willebrand disease (VWD). Commercial collagen binding assays have the advantage of immediate use for fast results, but are expensive. In this study we evaluated an in-house collagen-binding assay using type III collagen. We included it in the diagnostic work-up of 44 patients with VWD and 40 normal subjects. Other assays included VWF antigen, ristocetin cofactor activity, ristocetin-induced platelet agglutination and VWF multimeric analysis. The cost of this collagen-binding assay is 10-fold lower than that of commercial kits. The intra- and inter-assay coefficients of variation were <8% and <9% for normal values, respectively, and the normal reference range varies between 51% and 143%. This assay is sensitive to large VWF multimer representation, with a mean collagen-binding activity/antigen level (CB/Ag) ratio of 0.18 and 0.45 for type 2A and type 2B VWD, respectively, indicating functional discordance. It correlates with the antigen levels in type 2M and type 1 VWD, with mean CB/Ag ratios of 1.1 and 1, respectively. Our cost-effective in-house collagen-binding assay produced reliable results. We recommend the use of this test together with the ristocetin cofactor test in the diagnostic work-up of VWD.