Platelet vWF Research Articles

The effect of shear stress on the size, structure, and function of human von Willebrand factor.

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

Factors Modifying Risk of Bleeding in Patients with Continuous Flow Left Ventricular Assist Devices

<h3>Purpose</h3> Bleeding is a common problem in continuous flow left ventricular assist device (CF-LVAD) recipients. CF-LVAD patients can develop von Willebrand factor (vWF) dysfunction, however specific predictors of bleeding have not been elucidated. We hypothesized that vWF function, pump parameters, and platelet dysfunction are important risk factors for bleeding. <h3>Methods and Materials</h3> vWF analysis was performed before, and at multiple time points after device implant in 25 patients enrolled in the Vanderbilt Main Heart Registry who received a HeartMate II CF-LVAD. Platelet function (PFA-100) and VWF multimeric analyses were performed. <h3>Results</h3> All patients (84% male; 28% bleeders) had a reduction in high molecular weight multimers post-implant compared to pre-implant (•) (N - normal pooled plasma). Multimer reduction was similar in bleeders (°) and non-bleeders. [figure 1] Ristocetin cofactor activity was 30% lower than vWF antigen, and PFA-100 ADP closure times were significantly prolonged for all patients post-implant, consistent with the abnormal vWF multimer pattern. Average pump speed was higher in bleeders at all time points. <h3>Conclusions</h3> The development of vWF and platelet dysfunction in conjunction with higher pump speeds may contribute to bleeding events in CF-LVAD recipients. Monitoring these hemostatic parameters may allow us to identify patients prone to bleeding.

Specific Binding of Paraprotein to Platelet Receptors as a Cause of Platelet Dysfunction in Monoclonal Gammopathies

The study included 48 untreated patients with monoclonal gammopathies (MG). Paraprotein was isolated from the serum of 10 patients with decreased platelet aggregation. Platelet aggregation was measured before and after the addition of the isolated paraprotein to platelet-rich plasma (PRP) from 10 healthy donors, in vitro. Expression of platelet von Willebrand factor (vWF) receptor glycoprotein (GP)Ib and platelet collagen receptor GPVI was determined by flow cytometry in the PRP of healthy donors before and after the addition of isolated paraprotein using the monoclonal antibodies, CD42b (for GPIb) and CD36 (for GPVI). Flowcytometry showed that expression of CD42b and CD36 positive cells was reduced after the addition of isolated paraprotein to PRP from healthy donors (p < 0.001). These investigations demonstrated that paraprotein causes platelet dysfunction in patients with MG due to specific binding to the platelet vWF receptor GPIb and platelet collagen receptor GPVI.

Coagulation activity in renal ANCA-positive vasculitis

Two different methods (using Triton X-100 and glycerol) for lysing platelets to measure platelet vWF concentrations were compared directly. The platelet concentration of von Willebrand factor antigen (vWF:Ag) was similar for both methods, whereas ristocetin cofactor activity (Ricof) was higher with Triton than with glycerol. After storing platelet lysates for two months at −80°C vWF:Ag and Ricof concentrations decreased with both methods of lysis. Larger than normal (supranormal) vWF multimeric forms could be visualized in platelet lysates obtained using both methods, with no change of the multimeric pattern during storage. Triton can be recommended as the agent of choice to lyse platelets for measurement of their vWF concentration, but the samples must be assayed within two weeks to avoid decay of Ricof activity.

Direct Observation of von Willebrand Factor Elongation and Fiber Formation on Collagen During Acute Whole Blood Exposure to Pathological Flow

In severe stenosis, von Willebrand factor (vWF) experiences millisecond exposures to pathological wall shear rates (γ(w)). We sought to evaluate the deposition of vWF onto collagen surfaces under flow in these environments. Distinct from viscometry experiments that last many seconds, we deployed microfluidic devices for single-pass perfusion of whole blood or platelet-free plasma over fibrillar type 1 collagen (<50 ms transit time) at pathological γ(w) or spatial wall shear rate gradients (grad γ(w)). Using fluorescent anti-vWF, long thick vWF fibers (>20 μm) bound to collagen were visualized at constant γ(w)>30000 s(-1) during perfusion of platelet-free plasma, a process enhanced by EDTA. Rapid acceleration or deceleration of EDTA platelet-free plasma at grad γ(w)=±1.1×10(5) to ±4.3×10(7) s(-1)/cm did not promote vWF deposition. At 19400 s(-1), EDTA blood perfusion resulted in rolling vWF-platelet nets, although blood perfusion (normal Ca(2+)) generated large vWF/platelet deposits that repeatedly embolized and were blocked by anti-glycoprotein Ib or the α(IIb)β(3) inhibitor GR144053 and did not require grad γ(w). Blood perfusion at venous shear rate (200 s(-1)) produced a stable platelet deposit that was a substrate for massive but unstable vWF-platelet aggregates when flow was increased to 7800 s(-1). Triggered by collagen and enhanced by platelet glycoprotein Ib and α(IIb)β(3), vWF fiber formation occurred during acute exposures to pathological γ(w) and did not require gradients in wall shear rate.

Evidence of Selective Packaging and Different α-Granule Subtypes in Canine Platelets

Platelets are circulating megakaryocytic cytoplasmic fragments that are required to maintain vascular integrity and prevent haemorrhage. During activation, platelets release hundreds of bioactive proteins, mainly by secretion of α-granule content. These proteins include von Willebrand factor (vWF) and fibrinogen, major adhesive proteins involved in haemostasis. Human and mouse platelet α-granules are packaged selectively to contain different molecules and platelets can differentially release these proteins on specific receptor activation. Confocal laser scanning microscopy was used to analyse vWF and fibrinogen localization within canine platelet α-granules. In resting canine platelets, the majority of platelet vWF and fibrinogen is located in separate α-granule populations. These findings provide evidence that canine platelets contain different α-granule subtypes, suggesting that selective protein packaging occurs during canine platelet ontology.

Specific N- and O-Linked Carbohydrate Structures Mediate Von Willebrand Factor Interaction with Galectins -1 and -3

Abstract 2234Von Willebrand Factor (VWF) is extensively glycosylated with both N- and O-linked carbohydrates. Moreover, these complex glycan structures influence VWF functional properties, including susceptibility to ADAMTS13 proteolysis, and plasma clearance. The molecular mechanisms through which VWF glycosylation (including ABO blood group antigens) act to influence VWF physiology remains unexplained. However, recent data suggest that VWF circulates in normal plasma bound to various carbohydrate-binding proteins, including specific members of the galectin family. In addition, galectin-3 binding has been reported to influence VWF cleavage by ADAMTS13. In this context, we sought to elucidate the role of specific VWF glycan determinants in modulating galectin interaction.VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. VWF glycosylation was then modified using exoglycosidases and quantified by specific lectin ELISAs. Blood group specific VWF was also purified from pooled group AB, O, or Bombay plasmas. Galectins-1 and -3 were transiently expressed in competent E-coli cells with an N-terminal histidine tag, and purified by nickel chromatography. Finally, binding interactions were characterized via modified immunosorbant assay.In keeping with the previous report of Lenting et al, human pdVWF bound to both galectin-1 and galectin-3 in a dose-dependent manner. Enzymatic desialylation of pdVWF with α2-3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced binding to galectin-1 (231±6%, p<0.0001). Similarly, removal of terminal sialic acid also increased binding to galectin-3, albeit to a lesser extent (136±6%, p<0.05). To further define the role of VWF glycans in regulating galectin binding, pdVWF was exposed to sequential neuraminidase and galactosidase digestions to remove terminal sialic acid and sub-terminal galactose residues (NeuGal-VWF). In contrast to the enhanced binding of Neu-VWF, binding of NeuGal-VWF to both galectin -1 and -3 was significantly reduced (51±5% and 52±6% compared to pdVWF; p<0.005). Cumulatively these findings suggest that loss of capping sialic acid and exposure of sub-terminal galactose critically regulates VWF-galectin binding. Treatment with PNGase F to completely remove N-linked carbohydrate structures (PNG-VWF) markedly decreased binding to galectin -1 and -3 (13±1% and 57±2%, p<0.001). Moreover, combined PNGase F and O-glycosidase digestions further attenuated galectin-3 binding (21±1%, p<0.001), suggesting that both the N- and O-linked glycans are involved in mediating the VWF-galectin interaction.ABO(H) blood group antigens are expressed on both the N-linked and O-linked glycans of human VWF. Moreover, ABO(H) determinants influence VWF susceptibility to ADAMTS13 proteolysis and plasma VWF half-life, through unknown mechanisms. Purified VWF from normal group AB individuals bound to both galectin-1 and galectin-3 significantly better than group O VWF (146±8% and 483±19%; p<0.01). Conversely, no significant difference in binding was observed between Group O and Bombay VWF. Consequently, although terminal A (GalNAc) and B (Gal) sugar moieties promote galectin binding, expression of terminal α1–2 fucose residues is not important. The glycosylation profile of platelet-VWF differs from that of pdVWF. In particular, platelet-VWF expresses reduced levels of both capping sialic acid and sub-terminal galactose residues (∼50%), and lacks AB blood group antigens. To characterize the effects of this differential sugar expression on galectin binding, platelet-derived VWF was isolated and purified (platelet freeze-thawing followed by immuno-affinity chromatography with monoclonal CLB-Rag20). In keeping with the reduction in Gal and AB blood group antigen expression, platelet VWF bound less well to galectin-1 and galectin-3 (72±6% and 67±7% versus pdVWF; p<0.05).These novel data demonstrate that both the N- and O-linked oligosaccharide structures of VWF are involved in mediating galectin binding. In particular, expression of terminal AB blood group antigens, and expression of sub-terminal galactose moieties following loss of capping sialic acid, both markedly enhance galectin binding affinity. Further studies will be required to define how galectin binding is involved in mediating the functional consequences of variation in VWF glycans. Disclosures:No relevant conflicts of interest to declare.

Targeting FVIII Expression to Human Platelets Corrects the Hemophilic Phenotype in An Immunocompromised Hemophilia A Mouse Model Transplanted with Genetically Manipulated Human Cord Blood Stem Cells

Abstract 20Hemophilia A is a strong candidate for gene therapy because the therapeutic window is broad and a low plasma concentration of FVIII is therapeutic. However, the results of clinical trials so far have been disappointing due to either low expression or immune response problems. Our previous studies have demonstrated that lentivirus-mediated platelet-specific expression of FVIII (2bF8) in mouse platelets can ameliorate the bleeding phenotype in murine hemophilia A even in the presence of inhibitory antibodies. Since our ultimate goal is to express FVIII in human hemophilia patients’ platelets, we wanted to explore whether lentivirus-mediated gene transfer can efficiently introduce therapeutic levels of FVIII in human platelets and whether this human platelet-derived FVIII can correct the hemophilic phenotype. Human platelet-derived FVIII expression was introduced by 2bF8 lentivirus (LV)-mediated transduction of human cord blood (hCB) CD34+ cells followed by xeno-transplantation (hCBT) into immunocompromised NOD-scid IL2r-gamma knockout (NSG) mice. After bone marrow reconstitution, recipients were analyzed by flow cytometry, PCR, platelet lysate FVIII:C assay, immunogold-staining electron microscopy (EM), and non-restrictive LAM-PCR (nrLAM-PCR). About 30–67% of platelets in NSG/CBT mice were of human origin 3 weeks after transplantation, dropping to 3–18% at 5 weeks when 47–98% white blood cells (WBC) were of human origin. Expression of the 2bF8 protein product was detected in all recipients that received 2bF8 LV-transduced hCB cells, indicating viable engraftment of human CD34+ cells that were genetically modified with the 2bF8 LV transfer vector. Functional platelet-FVIII activity levels in the transduced mice were 0.74 ± 0.45 mU/108 total platelets (n = 6), which corresponded to 17.77 ± 10.36 mU/108 human platelets when converted to human platelets according to chimerism levels measured by FACS. EM determined that transgenic FVIII was colocalized with endogenous VWF in 2bF8 LV-transduced human platelets from hCBT recipients. nrLAM-PCR analysis showed that no identified insertion sites of 2bF8 LV were located in areas listed in the Retrovirus Tagged Proto-oncogene in human hematopoiesis database. To explore whether 2bF8 LV-transduced human platelets can ameliorate the bleeding phenotype of hemophilia A, we generated an immunocompromised hemophilia A mouse (SG/FVIIInull) model by crossing NSG mice into FVIIInull background and xeno-transplanted 2bF8 LV-transduced hCB CD34+ cells into these mice. We found that human platelet chimerism in recipients varied markedly with loss of NOD phenotype during breeding, while WBC chimerism was not affected. PCR analysis of 2bF8 proviral DNA confirmed viable 2bF8 LV-transduced human hematopoietic engraftment. FVIII:C was detected in platelet lysates with levels of 0.57 ± 0.41 mU/108 total platelets (18.30 ± 9.65 mU/108 human platelets (n = 11)). Four weeks after transplantation, all recipients (n = 7) that received 2bF8 LV-transduced hCB CD34+ cells survived tail clipping if animals had greater than 2% (3–57%) of platelets derived from 2bF8 LV-transduced hCB CD34+ cells, while 4 of 6 survived when human platelets were between 0.3% and 2%. Reduced survival in the latter group is presumably due to the limited number of human platelets containing FVIII. Rotational Thromboelastometry (TEG-like) analysis of whole blood confirmed that hemostasis was improved in SG/FVIIInull mice that received 2bF8 LV-transduced hCB CD34+ cells. Our results demonstrate that 2bF8 LV can efficiently introduce FVIII expression in human platelets and that human platelet-derived FVIII can improve hemostasis in hemophilia A mice, indicating that lentivirus-mediated platelet-derived FVIII gene therapy may be a promising approach for the treatment of human patients with hemophilia A. Disclosures:No relevant conflicts of interest to declare.

Phase II Biologic Effects Trial of Recombinant Interleukin-11 (rhIL-11, Neumega) in Moderate or Mild Hemophilia A or Von Willebrand Disease Unable to Use DDAVP,

Abstract 3308▪▪This icon denotes a clinically relevant abstract Background:DDAVP is the treatment of choice for individuals with type 1 von Willebrand disease (VWD), although 20% are unresponsive, and of the 80% who do respond, the VWF increase is transient, as endothelial stores are depleted after 3 days. Further, administration requires a 30- minute intravenous infusion in a medical facility. Plasma-derived concentrates may be used in these settings, but are more costly and have potential risk of transmissible infection. We recently demonstrated that recombinant human IL-11 (rhIL-11, Neumega®), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, increases VWF activity up to 2-fold when given daily by subcutaneous injection, with levels persisting each day it is given, and reduces menstrual and postoperative bleeding. The effects of rhIL-11 in individuals with VWD unresponsive or allergic to DDAVP, or hemophilia A, however, have not been evaluated. Methods:We conducted a phase II trial to evaluate the safety and biologic effects of rhIL-11 in VWD patients unresponsive or allergic to DDAVP (VWD-Un) or mild hemophilia A (HemA). rhIL-11 was given subcutaneously at 25 μg/kg daily for 4 days in the non-bleeding state, followed on day 4, 30 minutes after rhIL-11, by one dose of DDAVP intravenously, 0.3 μg/kg, if not contraindicated (pt. 2). Fluid restriction was recommended. Fluid status was assessed by height, weight, and exam. Pre- and post-dosing laboratory assays included the VWD profile, VWF multimers by SDS gel electrophoresis, and platelet VWF mRNA by qPCR. Results:The results of the first six subjects, including three with VWD (one type IIB and two type 1 VWD), VWF:RCo 0.10–0.20 U/ml, and three with mild hemophilia A, F.VIII 0.08–0.12 U/ml, are presented. All subjects were healthy, with no hypertension or cardiac disease, and all had normal physical exams and normal EKGs. By day 4, among VWD-Un subjects, there was a 1.2-fold increase in VWF:RCo (15±3% vs. 12±0%); a 1.6-fold increase in VWF:Ag (22±8% vs.14±6%); and a 1.3-fold increase in VIII:C (34±36% vs. 27±10%), as compared with pre-rhIL-11 levels (Figure). Following DDAVP (except pt. 2), there was an additional 2.0-fold, 1.7-fold, and 2.6-fold increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. Among HemA subjects, by day 4, there was a 1.8-fold increase in VWF:RCo (160±25% vs. 88±12%); a 1.8-fold increase in VWF:Ag (182±28% vs.99±18%), p<0.01; and a 1.5-fold increase in VIII:C (21±8% vs. 14±5%), as compared with pre-rhIL-11 levels. Following DDAVP, there was an additional 1.5-fold (p<0.01), 1.7-fold, and 2.8-fold (p<0.05) increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. The drug was well tolerated well with less than grade 1 mild conjunctival erythema, local erythema and tenderness at the injection site; in one subject transient hyponatremia, Na 129 meq/L, occurred after excess oral fluid intake for diabetic hyperglycemia, which resolved with fluid restriction. Discussion:These data suggest that rhIL-11 increases VWF and VIII levels modestly in VWD patients unresponsive/allergic to DDAVP, and in mild hemophilia A, suggesting the potential use in treatment of clinical bleeding in these disorders.SubjectsDiagnosisVWF:RCo(%)VWF:Ag(%)VIII:C(%)PreDay4DDAVPPreDay4DDAVPPreDay4DDAVP1. 25/FI-VWD12±021±945±2612±525±6*49±1637±1810±6125±36*2. 22/FIIBVWD12±012±0ND25±335±18ND37±1286±13†ND3. 25/FI-VWD12±012±032±0*4±06±0*26±157±05±0*54±344. 25/MHemA78±5121±25227±11*81±7139±3*207±6*10±019±0*78±12*5. 31/MHemA73±3151±43191±2580±6171±9*222±20*25±137±174±4*6. 51/MHemA112±42208±0*324±60135±3235±13*273±44*7±08±0*24±8**p< 0.001;†p< 0.01. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: Implication for the interpretation of satellite bands

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes.

Regulation der primären Hämostase durch von-Willebrand-Faktor und ADAMTS13

Von Willebrand factor (VWF) is an adhesive, multi-functional huge multimerized protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and coagulation factor VIII (FVIII). The functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind and protect FVIII, an important cofactor of the coagulation cascade. VWF function in primary haemostasis is located in particular in the arterial and micro-circulation. This environment is exposed to high shear forces with hydrodynamic shear rates ranging over several orders of magnitude from 10⁻¹ to 10⁵ s-1 and requires particular mechanisms to enable platelet adhesion and aggregation under these variable conditions. The respective VWF function is strictly correlating with its multimer size. Lack or reduction of large VWF multimers is seen in patients with von Willebrand disease (VWD) type 2A which correlates with reduction of both VWF:platelet GPIb-binding and VWF:collagen binding and a bleeding phenotype. To prevent unlimited platelet adhesion and aggregation which is the cause of the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), VWF function is regulated by its specific protease ADAMTS13. Whereas a particular susceptibility of VWF to ADAMTS13 proteolysis is the cause of a frequent VWD type 2A phenotype, lack or dysfunction of ADAMTS13, either acquired by ADAMTS13 antibodies or by inherited ADAMTS13 deficiency (Upshaw-Schulman Syndrome), causes TTP. Therefore VWD and TTP represent the opposite manifestations of VWF related disorders, tightly linked to each other.

Phase II prospective open-label trial of recombinant interleukin-11 in women with mild von Willebrand disease and refractory menorrhagia

Lack of effective treatment for menorrhagia is the greatest unmet healthcare need in women with von Willebrand disease (VWD). We conducted a single-centre phase II clinical trial to determine efficacy and safety of recombinant IL-11 (rhIL-11, Neumega®) given subcutaneously for up to seven days during six consecutive menstrual cycles each in seven women with mild VWD and menorrhagia refractory to haemostatic or hormonal agents. rhIL-11 reduced menstrual bleeding severity as measured by pictorial blood assessment chart (PBAC) ≥ 50% (to <100) in 71% of subjects, cycle severity ≥ 50% in 71%, and bleeding duration ≥ 2 days in 85%, all p ≤ 0.01. After rhIL-11, plasma VWF:RCo increased 1.1-fold, but did not correlate with PBAC, r=0.116, bleeding duration, r=0.318, or cycle severity, r=-0.295, or hsCRP, r=-0.003, all p>0.05. Platelet VWF mRNA expression by quantitative PCR increased mean four-fold (1.0-13.5). rhIL-11 was well tolerated with grade 1 or less fluid retention, flushing, conjunctival erythema, and local bruising. In summary, rhIL-11 reduces menorrhagia safely and warrants further study.

Comparative Analysis of von Willebrand Factor Profiles in Pulsatile and Continuous Left Ventricular Assist Device Recipients

A higher rate of nonsurgical bleeding has been observed in nonpulsatile left ventricular assist device (LVAD) recipients. von Willebrand factor (vWF) profiles were compared for nonpulsatile and pulsatile LVAD recipients to explore mechanisms that may contribute to the development of postimplant nonsurgical bleeding. The nonpulsatile mechanism may impair vWF function by creating a deficiency in vWF high molecular weight multimers (HMWMs), essential for hemostasis. High molecular weight multimer deficiency should result in low ristocetin cofactor (RCo) to vWF antigen ratios (vWF:RCo/vWF:Ag) because of impaired platelet (plt)-binding ability. von Willebrand factor profiles and HMWM were measured pre- and post-LVAD placement in 11 nonpulsatile (HeartMate II [HM II[) and 3 pulsatile (HeartMate XVE [HM XVE]) recipients. All the nonpulsatile LVAD recipients exhibited loss of HMWM 30 days postimplant. The vWF:RCo/vWF:Ag ratio was significantly lower after LVAD placement in the nonpulsatile group when compared with the pulsatile group. In addition, the vWF:RCo/vWF:Ag ratio decreased significantly from baseline 30 days postimplant within the nonpulsatile recipients. All nonpulsatile LVAD recipients had low vWF:RCo/vWF:Ag ratios 30 days post-LVAD even if the values were normal at baseline. These data suggest that nonpulsatile HM II recipients develop HMWM loss and impaired vWF platelet (plt)-binding ability after LVAD placement. Similar results were not observed in our small series of pulsatile HM XVE recipients. This finding could suggest a contributing factor to the increase in nonsurgical bleeding observed in nonpulsatile LVAD patients. Further investigation is ongoing to identify specific causes of vWF impairment.

The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset of vessels in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet alpha-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention.

Platelet vWF, ADAMTS-13, and Neutrophil Activation in Patients with Essential Thrombocythemia (ET)

ET is a chronic myeloproliferative disorder, the benign clinical course of which can be complicated by both thromboses and hemorrhages. The levels of the largest multimers of plasma vWF, are relevant for the hemostatic balance in this disease. vWF is stored in the cellular granules of both endothelial cells and platelets, from which is released upon activation. Neutrophils and platelets circulate in an activation status in ET patients, leading to an increased formation of platelet/neutrophil aggregates. In this study we evaluated in ET patients, the platelet vWF content and its relation to neutrophil activation. In addition, the activity of ADAMTS13, the enzyme responsible for maintaining the normal size distribution of vWF multimers, was studied. A group of 61 consecutive ET patients (50% carrying the JAK2 V617F mutation) and 30 healthy controls were included into the study. Total vWF antigen (vWF:Ag) and activity (vWF:Act) levels were determined in both plasma and in isolated washed platelets by ELISA and collagen binding assay (CBA), respectively. ADAMTS13 activity was measured in plasma according to Gerritsen's method. CD11b and leukocyte alkaline phosphatase (LAP) were measured on neutrophil membrane as activation markers. Plasma vWF:Ag levels resulted significantly increased in ET patients compared to controls (p<0.05), the subgroup of patients with the JAK2 mutation showing the highest levels; whereas vWF:Act was not statistically different. The vWF Act/Ag ratio was significantly lower in ET compared to controls (0.85±0.15 vs 0.95±0.23; p<0.01). A significant inverse relation between platelet count and plasma vWF:Act existed. On the other hand, platelets from these patients had a decreased content of both vWF:Ag and vWF:Act than platelets form controls (p<0.01), suggesting an increased platelet release of vWF. In addition, platelets from ET JAK2 mutation carriers had significantly lower vWF:Act compared to wild-type subjects. Accordingly, the platelet vWF Act/Ag ratio of JAK2 mutation carriers was significantly (p<0.01) lower vs wild-type patients and controls. Neutrophil confirmed to be activated in these patients, as previously described. Indeed, surface activation markers were increased in ET patients compared to controls, and, in particular, CD11b levels were inversely related to platelet vWF:Act (p<0.01). The measurement of plasma ADAMTS13 showed a reduction in the activity of this protease in ET patients (68±16%) compared to normal control mean (p<0.01). In conclusion, in ET patients and particularly in JAK2 mutation carriers, a decrease in platelet vWF:Ag and Act is found and correlates with neutrophil activation, suggesting a role of neutrophil in platelet degranulation. Increased vWF release and reduced ADAMTS13 activity can represent a mechanism of hypercoagulation in these patients.

Platelet GPIbα, GPIV and vWF polymorphisms and fatal pre-hospital MI among middle-aged men

The binding of platelet glycoprotein (GP) Ib-IX-V receptor complex to subendothelial collagen via von Willebrand factor is the initial step of the formation of platelet thrombi following atherosclerotic plaque rupture. Platelet GPIV binds to collagen and/or thrombospondin and further activates platelets. Genetic variation in these proteins could associate with platelet aggregability and the risk of myocardial infarction (MI). We studied the associations of polymorphisms of GPIbalpha, GPIV and von Willebrand factor with the extent of coronary atherosclerosis, coronary narrowing, and fatal MI in an autopsy series of 300 middle-aged, Caucasian Finnish men who had suffered sudden out-of-hospital death. 31% of men with MI under the age of 50 carried the GPIbalpha HPA-2 ThrThr/Kozak TT haplotype as opposed to 62% of control men (OR 0.27, 95% CI 0.08-0.93, P = 0.03). In addition, 7% of men with MI under the age of 50 carried the GPIV AA genotype versus 29% of control men (OR 0.16, 95% CI 0.03-0.98, P < 0.05). These associations were not due to any effects of these gene variants on the coronary atherosclerotic changes. The G/A polymorphism of the von Willebrand factor gene failed to show any association with MI or coronary atherosclerosis in this series of men. The combined ThrThr/TT haplotype of GPIbalpha as well as the AA genotype of GPIV seem to decrease the risk of fatal MI among men during early middle-age.

Formation of Platelet Strings and Microthrombi in the Presence of ADAMTS13 Inhibitor Does Not Require P-Selectin or β3 Integrin.

VWF is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies respectively. VWF released from the storage granules upon cellular activation or stimulation with secretagogues are extremely large and known as Ultra-Large von Willebrand factor (UL-VWF). Such filaments were first shown by Dong and colleagues (Dong et. al., 2002) and platelets were seen to align as string of beads on these UL-VWF multimers. These platelet strings are then rapidly cleaved to smaller size by the metalloprotease ADAMTS-13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13). Recently, we have similarly shown that in Adamts13−/− mice platelet strings form and remain on stimulated endothelium in veins, where they wave in the blood stream for many minutes (Motto et. al., 2005). We have also shown that antibody to ADAMTS13 induces platelet strings in activated venules and transient thrombi formation in activated microvenules of wild type similar to Adamts13−/− mice (Chauhan et. al., 2006). The obvious question is what anchors the UL-VWF mulimers to the endothelial surface? In a flow chamber model, using polyclonal antibodies to P-selectin or soluble P-selectin, it was proposed that P-selectin anchors the UL-VWF multimers to the endothelial surface. The present study was designed to address the role of P-selectin and that of the platelet and endothelial integrin β3 in platelet string formation in vivo using intravital microscopy and genetically engineered mice. We demonstrate that in histamine activated venules, causing Weibel-Palade bodies secretion, P-selectin is not required for formation or retention of VWF platelet strings on endothelium in vivo. The mean time the platelet strings (~30–100 μm long) anchored onto the endothelium was 45 s. This duration was significantly longer than in P-selectin−/− mice infused with control Ig (P<0.001) and similar to WT mice treated with the inhibitory antibody. Similar to wild type, 45 s after topical superfusion of calcium ionophore A23187, platelet strings forming transient thrombi formation was seen in microvenules (25–30 μm in diameter) of P-selectin−/− mice when the ADAMTS13 activity was inhibited. Platelet strings were either not seen or were very short lived in the β3−/− mice infused with control Ig, whereas in the mice infused with anti- ADAMTS13 antibody platelet strings could be seen that anchored to the endothelium with a mean time of 50 s (P<0.0001). These observations show that platelet strings are not held by β3 integrin and platelets lacking β3 integrin can adhere firmly to the VWF strings in the histamine activated venules. VWF containing platelet strings could also entangle and form emboli in the absence of β3 integrin.

Laboratory Testing for von Willebrand Disease: Contribution of Multimer Analysis to Diagnosis and Classification

The stepwise diagnosis of von Willebrand disease (vWD) includes patient and family history, screening procedures (bleeding time, filter tests, platelet counts, activated partial thromboplastin time [aPTT]), confirmatory tests (von Willebrand factor [vWF]:antigen [Ag], vWF:ristocetin cofactor activity assay [RCo], vWF:collagen-binding test [CB], ristocetin-induced platelet agglutination [RIPA], and factor [F] VIII:coagulant activity [C]) and tests for final classification (multimeric analysis, vWF:factor VIII binding, and platelet vWF). Accumulating knowledge of the different clinical phenotypes and the pathophysiological basis of the disease have been translated into a classification that differentiates between quantitative and qualitative defects by means of quantitative and functional parameters and by analyzing the electrophoretic pattern of vWF multimers, but without inclusion of the genotype. Recently, it has been shown that with a sensitive method of multimer analysis, a > 90% genotype-phenotype relation may be achieved in the near future.

Platelet-Derived Factor VIII (FVIII) Corrects the Murine Hemophilia A Phenotype Even in the Presence of FVIII Inhibitors.

Inhibitory antibody development is not only the most severe and important complication of FVIII infusion in hemophilia A patients, but also a major potential concern in patients following hemophilia A gene therapy. We have developed a transgenic model in which human B-domain deleted FVIII (hFVIII) expression is controlled by the platelet-specific αIIb promoter (2bF8) and results in FVIII storage together with VWF in platelets. The transgenic mice were then bred into FVIII knockout mice (FVIIInull) so that the only FVIII in peripheral blood is derived in platelets. The bleeding phenotype was corrected in the 2bF8/FVIIInull transgenic mice, even though FVIII was not detectable in plasma. We further hypothesize that platelet-derived FVIII in a complex with VWF will 1) protect FVIII from inactivation by circulating FVIII-specific antibodies and 2) release FVIII in sufficiently high local concentration at sites of vessel damage so as to overcome immediate inactivation by FVIII inhibitors. To address our hypothesis, we used three strategies -1) an acute model infusing plasma from highly immunized FVIIInull mice, 2) immunization of 2bF8 transgenic mice with human FVIII, and 3) a model using transplantation of spleen cells from highly immunized mice into sub-lethally irradiated 2bF8 transgenic mice. Thirteen mice were infused with plasma containing 48-64 BU and 12 survived following tail clipping using a 1.59 mm template. FVIII-immunized transgenic mice (given adjuvant) generated anti-FVIII inhibitory antibodies as high as 350 BU after a single immunization. The level of platelet-FVIII and the platelet number in these mice were not significantly different from pre-immunization levels and 13 of 15 survived tail clipping. In the 3rd group, spleen cell transplantation resulted it titers of >5000 BU and 8 of 12 transgenic mice survived tail clip. In contrast, only 2 of 7 wild type mice survived tail clip with inhibitor titers >5000 BU after spleen cell transplantation. None of 9 untransplanted FVIIInull mice survived. These results demonstrate that platelet-derived FVIII is protected from inhibitor inactivation and correct the phenotype of murine hemophilia A even in the presence of a high-titer anti-FVIII antibody. This approach may be useful not only for preventing inhibitor formation in hemophilia A gene therapy but also for gene therapy in the case of pre-existing inhibitors.

The Metalloprotease ADAMTS13 Is a Natural Anti-Thrombotic.

The metalloprotease ADAMTS13 cleaves highly adhesive ultra large von Willebrand factor (UL-VWF) multimers after their release from the endothelium. UL-VWF is stored in Weibel-Palade bodies of endothelial cells. ADAMTS13 deficiency is linked to a life threatening disorder, thrombotic thrombocytopenic purpura (TTP), which is characterized by VWF and platelet-rich thrombi in the microvasculature, and may lead to organ ischemia, neurological dysfunction and ultimately death. We previously confirmed in vivo that in Adamts13−/− mice and not in wild-type mice “strings of platelets” formed after endothelial activation. The VWF strings remain attached at one end to the endothelium and “waved” the other end in the blood stream. In addition, we showed prolonged adhesion of platelets to secreted VWF on endothelium of Adamts13−/− mice. The possible role of ADAMTS13 in platelet plug formation has not been addressed previously. We investigated thrombosis in venules and arterioles of Adamts13−/− mice by intravital microscopy. Mesenteric microvenules were stimulated with calcium ionophore A23187, a secretagouge of Weibel-Palade bodies. In the Adamts13−/− mice we observed spontaneous thrombus formation in the activated microvenules. Inhibitory antibody to plasma ADAMTS13 induced thrombi formation in the activated microvenules and prolonged adhesion of platelets to secreted VWF on endothelium of wild-type mice. In the ferric chloride arterial injury model we found that the number of fluorescent platelets deposited within 2–3 min on the sub-endothelium after injury was unexpectedly higher in Adamts13−/− mice as compared to Adamts13+/+ mice (P<0.05). In the Adamts13−/− mice, platelet plug grew faster as thrombi >30 um were seen at 6.6±0.9 min compared to 10.8±0.8 min in the Adamts13+/+ mice (P<0.005). The platelet plug reached occlusive size in 10.6±0.7 min in Adamts13−/− mice, whereas in the Adamts13+/+ mice all the vessels were still open at this time. In the Adamts13+/+, the mean vessel occlusion time was 16.7±1.2 min after injury (P<0.0005). Thus ADAMTS13, by cleaving VWF multimers, down regulates both platelet adhesion to exposed subendothelium and thrombus growth. Furthermore, we observed that recombinant human ADAMTS13 inhibited formation of VWF platelet strings in histamine-activated venules and promoted thrombi dissolution in the Adamts13−/− mice. Our findings reveal that ADAMTS13 has natural anti-thrombotic activity and recombinant ADAMTS13 could be used as an anti-thrombotic agent.