vWF Multimeric Composition Research Articles

Reduced Cleavage of Von Willebrand Factor By ADAMTS13 Exacerbates Acute Kidney Injury Secondary to Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) antigen levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated following severe injury. We hypothesized that following traumatic injury, endothelial damage and activation would result in the release of high molecular weight multimeric (HMWM) vWF forms into circulation with a concominant decrease in ADAMTS13. We further hypothesized that the alterations in circulating levels and functions of these factors, following traumatic injury, promote an increased thrombogenic and inflammatory response, which exacerbates the risk of developing post traumatic acute kidney injury (AKI). Methods: Plasma samples from a cohort of patients with trauma induced AKI, collected at 0-, 24-, and 72-hours after admission, were evaluated against plasma samples from trauma patients that were matched for injury characteristics, but were without AKI. VWF and ADAMTS13 activity and antigen levels were evaluated. Circulating vWF multimeric composition was determined by vertical agarose gel electrophoresis. We further evaluated the effects of traumatic injury on ADAMTS13 and vWF by means of a pre-clinical model of trauma and hemorrhage, which is comprised of a liver laceration, cardiac puncture, and soft tissue injury. Following this polytrauma model, hepatic ADAMTS13 RNA expression was evaluated by qRT-PCR of total liver RNA; analyzed liver samples were taken immediately before, 24-, and 72-hours after injury. The impact of the polytrauma model on renal function was evaluated via changes in plasma cystatin C, while the effect on the renal microvasculature was evaluated by means of immunofluorescent staining of vWF and fibrinogen in kidney sections. Results: VWF antigen was increased in trauma patients when compared to healthy controls upon admission (11.0 ± 3.72 ug/mL vs. 4.64 ± 0.91 ug/mL), persisting through 72-hours (13.11 ± 3.16 ug/mL). The percentage of HMWM vWF forms in trauma patient samples was elevated at all time points compared to pooled normal plasma (PNP) (27.9 ± 4.5% and 15.9 ± 0.8). Trauma patient ADAMTS13 activity was decreased at all time points (0.48 ± 0.14 IU/mL vs. 1.27 ± 0.26 IU/mL). AKI patients showed a 1.54 ± 0.48-fold increase in vWF antigen levels over the 72-hour period (p=0.016), while there was no change in the control trauma group. Those who developed AKI demonstrated a larger proportion of HMWM vWF at 72-hours than their non-AKI counterparts (32.9 ± 2.4% vs 28.8 ± 3.2%). qRT-PCR of murine total liver RNA, following the polytrauma model, exhibited ADAMTS13 RNA expression levels that were 35% of normal at 24-hours and 60% of normal at 72 hours post-injury. A matched analysis of murine plasma cystatin C levels demonstrated a marked elevation 24-hours post polytrauma model (sham: 510ng/mL (435-561) vs trauma: 550 (479-701)). H&E staining of kidney sections, which were harvested following the polytrauma model, demonstrated generalized renal tubule dilation and denudation, suggestive of post-traumatic acute tubular necrosis. Immunofluorescent studies of kidney sections from the same mice exhibited 8-15µm microvascular plugs that were positive for fibrinogen with a vWF core; renal immunofluorescent studies of control mice demonstrated no microvascular depositions. Conclusions: These data indicate that traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes inflammation and thrombosis. In the immediate moments following injury, these mechanisms support life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to a burden that may produce microvascular clot deposition, exacerbating pathologic states such as acute kidney injury. Disclosures No relevant conflicts of interest to declare.

Prolonged Circulation of Ultra-Large Von Willebrand Factor and a Reduction in ADAMTS13 Activity Promotes Microvascular Disease Following Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF (UL-vWF) is released into the bloodstream by damaged endothelium, resulting in increased thrombogenicity due to circulating vWF multimers. We further hypothesized that traumatic injury would lead to a deficit of ADAMTS13, promoting the accumulation of UL-vWF forms and, ultimately, the increased risk of microvascular disease, such as acute kidney injury (AKI). Methods: A cohort of 37 severely injured trauma patients was analyzed for antigen levels of plasma vWF and ADAMTS13 at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis. Multivariate analyses were performed with data abstracted from the electronic medical records to identify further dependences. A similar analysis was also performed on plasma from a cohort of 8 patients with trauma induced AKI at 0-, 24-, and 72-hours after admission; these patients were well matched against trauma patients without AKI. Finally, we utilized a murine model of polytrauma and hemorrhage, in conjunction with qRT-PCR of ADAMTS13 in total liver RNA, to specifically address how the expression of ADAMTS13 is altered by the systemic effects of traumatic injury. Results: Circulating vWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultra-large vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%). The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma. Severe trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients. Furthermore, the proportion of circulating low molecular weight multimeric (LMWM) vWF to total circulating vWF forms was directly dependent upon ADAMTS13 activity at 24-hours (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) LMWM vWF vs Normal Activity: 25.8% (22.7-35.2) LMWM vWF). Strikingly, ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45). The cohort of 8 trauma patients who went on to develop AKI showed a 1.54-fold (1.02-2.05) increase in plasma vWF antigen levels between 0 and 72 hours, while those who did not develop AKI showed no change in vWF levels over this time period. Furthermore, those who developed AKI demonstrated a smaller proportion of LMWM vWF in plasma than those who did not (25.4% (23.4-28.0) vs 31.2% (27.2-35.6)), suggesting the increased thrombogenicity of their circulating vWF forms. Finally, qRT-PCR of total liver RNA in 6 mice demonstrated a 2-fold decrease in ADAMTS13 RNA expression levels between the times immediately before and 24-hours after trauma. Altogether, these data indicate that both circulating ADAMTS13 and its production are deficient in the days following severe injury. Conclusions: Severe traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes thrombosis. Not only is the concentration of circulating ADAMTS13 decreased following traumatic injury, but hepatic expression of the enzyme is lacking as well. In the immediate moments following injury, these mechanisms contribute to life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to burden that may exacerbate microvascular disease. Disclosures Ragni: Bioverativ/Sanofi: Consultancy, Research Funding; Sangamo: Research Funding; Shire/Takeda: Consultancy, Other: Study drug; Alnylam/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; Spark Therapeutics: Consultancy, Research Funding; ICER: Consultancy; OPKO: Research Funding; Biomarin: Consultancy, Research Funding. Rollins-Raval:Bayer, Inc: Membership on an entity's Board of Directors or advisory committees. Raval:Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer, Inc: Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Expression of terminal α2-6–linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13

Abstractvon Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (± 14%) by ADAMTS13, compared with 11% (± 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O ≥ B > A ≥ AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.

Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Abstract 30VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity.Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis.Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression.Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation.These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures:No relevant conflicts of interest to declare.

Molecular diagosis of von Willebrand disease.

von Willebrand disease (vWD) is one of the most common inherited human bleeding disorders, which is caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is a highly multimerized glycoprotein that promotes platelet adhesion and aggregation at a high shear rate, while also acting as a carrier of coagulation factor VIII. vWD has been subdivided into three categories, which reflect their pathophysiology. Type 1 and type 3 vWD reflect partial or complete deficiency of vWF, whereas type 2 vWD reflects qualitative defect of vWF. The ability of vWF to interact with its platelet receptor and factor VIII, and the analysis of the multimeric composition of vWF are essential to identify patients with different vWD subtypes. The prevalence of different vWD subtypes was reported in the literature. In the past years, ninety-one patients with vWD were consulted in our institution. Of all the vWD patients, 56 (61.5%) belong to type 1, 26 (28.6%) type 2 and 9 (9.89%) type 3. The analysis of vWF gene was performed in some type 2 and type 3 vWD by denature gradient gel electrophoresis and sequencing. We have found six cases of point mutations of vWF gene, Ala737-->Glu, Gly 22-->Glu, Met37 Val and Ser71-->stop codon. Substitutions, are first reported in international database. We constructed an expression plasmid pSVA737EvWF containing full length of cDNA of vWF which included the Ala737 Glu substitution by site-direct mutagenesis. The structure of recombinant vWF within transfected COS-7 cells and the secretion of high-molecular-weight (HMW) multimers were similar to wild-type vWF. HMW forms of vWF multimers were absent in plasma but present in platelets. The mutation corresponds to the group II type 2A vWD characterized by normal secretion of all vWF multimers.

Von Willebrand factor binding to heparin in various types of von Willebrand disease

The purpose was to study von Willebrand factor (vWF) binding to heparin in different types of von Willebrand disease (vWD). Plasma samples from 92 patients were representative of most vWD subtypes as they included 13 type 1, ten type 2N, 27 type 2A, 23 type 2B, and 19 type 2M patients. We selected assay conditions suitable for the screening of plasma vWF concentrations as low as 15 U/dl vWF:Ag. We determined the range of vWF concentrations in plasma where the percentage of (125)I-MAb/vWF complexes bound to heparin-agarose beads was constant. This range of dilution allowed circumvention of potential competition by other plasma heparin-binding proteins. The multimeric composition of vWF had hardly any influence on the ability of vWF to bind to heparin. Results were expressed as the ratio of heparin-binding capacity of patients' plasma to that of normal pool plasma. We found a ratio of 0.99+/-0.004 (mean+/-s.e.m.) for 23 normal individual donors. Furthermore, when comparing the mean values of plasma vWF-heparin binding ratios by ANOVA F-test in the six groups (one normal and five vWD), we found significant differences between them (P<0.0001). Pairwise comparison of multiples by the Scheffe's test indicated that the mean values of ratios in type 2A on the one hand and type 2M on the other, were significantly lower than in normal plasma, type 2N, type 2B and type 1. Our data suggest a relationship between the ability of vWF to bind to heparin and to the platelet GPIb receptor, since type 2B and 2N patients have an increased or normal ability to bind to GPIb whereas type 2A and 2M patients have an impaired interaction with that receptor.

Clinical guidelines for treating von Willebrand disease patients who are not candidates for DDAVP--a survey of European physicians.

While certain plasma-derived factor VIII/von Willebrand factor (FVIII/vWF) concentrates have proven to be quite useful in preventing or controlling bleeding in persons with von Willebrand disease who are not candidates for DDAVP, most of the information concerning dosage and effectiveness has been anecdotal. Additionally, the laboratory tests used to quantify vWF (the vWF:RCoF assay and collagen binding assay) are not well standardized. Thus, the US Food and Drug Administration (FDA) has been reluctant to grant licensed indication for use of these products in von Willebrand disease. This brief report describes a survey of non-US physicians (from major centres) who are recognized experts in haemophilia and von Willebrand disease. Twenty-four of 27 questionnaires were completed, returned and analysed. Products thought to be effective in von Willebrand disease included Haemate P, Facteur von Willebrand, Alphanate, and the UK's BPL '8Y'. In calculating dosage to stop or prevent bleeding, most aim for a certain level of both FVII and vWF:RCoF. Postoperatively, 16/24 respondents follow both of these laboratory tests once daily. Twenty-two of 24 would follow FVIII levels once daily. It is noteworthy that FVIII assay results are generally the only test results readily available to guide the respondents' clinical decisions. For treatment of significant mucous membrane bleeding, respondents often individualize dosage and monitoring, depending on the type, location and extent of bleeding. Gastrointestinal bleeding is generally treated more aggressively. Patient monitoring varies between merely looking for cessation of bleeding, to a battery of laboratory tests, including haemoglobin and haematocrit. Seven out of 24 would monitor BT as well. In addition to FVIII content, 22/24 respondents noted that they would find it helpful to have vWF:RCoF listed on the label, while 15/24 would like to know the vWF multimeric composition.

Changes in the aortic endothelium and plasma von Willebrand factor levels during the onset and progression of insulin-dependent diabetes in BB rats

Endothelial injury has been implicated in the enhanced vascular disease associated with diabetes mellitus. In diabetic humans elevated plasma von Willebrand factor (vWF) levels have been interpreted as an indication of endothelial damage. Using the BB rat as a model of inherited insulin dependent-diabetes mellitus, plasma vWF and aortic endothelial ultrastructural alterations were examined during the first 7 months of diabetes. Total plasma vWF levels were determined by ELISA and vWF multimeric composition by electrophoresis. vWF was identified immunohistochemically. Following the onset of hyperglycemia, there were progressive alterations in aortic endothelial morphology, which were consistent with injury, and aortic intimal thickening was significantly greater in rats diabetic for 7 months compared to age-matched controls. Significant increases in the Weibel Palade (WP) body content of the endothelial cells were observed after 1 week and 2 months of diabetes, but not at later times. Endothelial alterations associated with the possible release of vWF appeared to involve fusion of WP bodies with other vacuoles rather than direct fusion with the cell membrane. Plasma vWF levels in diabetic rats were varied, but were not significantly different from those of control animals and did not correlate with either glucose or insulin levels. The multimeric composition of plasma vWF was also similar at all times in both diabetic and non-diabetic animals. From these observations, plasma vWF levels do not provide an indicator of the endothelial perturbations which occurs in diabetic rats.

Severe, recessive von Willebrand’s disease in German Wirehaired Pointers

Objective To characterize the biochemical and genetic basis of a clinically severe form of von Willebrand’s disease (VWD) in German Wirehaired Pointers. Design Case series. Sample Population Plasma samples from 335 German Wirehaired Pointers (8 clinically affected). Procedure Plasma samples were evaluated, using an ELISA, to determine von Willebrand factor (WVF) antigen concentration. Additional studies performed on samples from clinically affected dogs included coagulation screening tests, factor VIlI coagulant activity assays, and immunoelectrophoresis to determine VWF multimeric composition. Results Mucosal bleeding and bleeding at sites of cutaneous injury were observed in affected dogs by 6 months old. Plasma vWF antigen concentration was markedly reduced, and there was a lack of high molecular weight vWF multimers; findings compatible with type-ll vWD. Inheritance and expression pattern of vWD in this breed was most compatible with that of an autosomal recessive trait. Clinical Implications Von Willebrand's disease should be included in the differential diagnoses of bleeding diatheses in German Wirehaired Pointers, with definitive diagnosis confirmed on the basis of canine-specific vWF assays. (J Am Vet Med Assoc 1996; 209:926-929)

Abnormally large von Willebrand factor multimers in Henoch‐Schönlein purpura

Allergic vasculitis phenomena seem to be involved in Henoch-Schönlein purpura (HSP). Elevated plasma levels of von Willebrand factor (vWf) are a well recognized feature of vasculitis and have been taken as an indication of in vivo endothelial cell damage. Plasma factor VIII:C and vWf levels and vWf multimeric pattern were studied in 8 patients with HSP, during active disease and twice during the remission (3 and 9 months later). Plasma vWf multimeric composition was evaluated using low resolution gels which better resolve large vWf multimers. During active disease plasma factor VIII:C, vWf:Ag, and vWf:RCoF were normal in 5% of patients and increased in three, but in each patient, platelets appeared to aggregate at doses of ristocetin lower than in normals. Furthermore, all patients demonstrated the presence of abnormally large vWf multimers usually found only in platelets and endothelial cells. Three and 9 months later, during remission, in spite of the normalization of factor VIII:C and vWf levels, the abnormal multimers were still detectable, as well as hyper-responsiveness to ristocetin. These findings confirm that damage and/or perturbation of endothelial cells is associated with HSP. Moreover, the persistence of abnormality in the vWf multimeric pattern, when the disease is inactive, suggests that the mechanisms involved operate through the entire clinical course. © 1996 Wiley-Liss, Inc.

Abnormally large von Willebrand factor multimers in Henoch-Schönlein purpura.

Allergic vasculitis phenomena seem to be involved in Henoch-Schönlein purpura (HSP). Elevated plasma levels of von Willebrand factor (vWf) are a well recognized feature of vasculitis and have been taken as an indication of in vivo endothelial cell damage. Plasma factor VIII:C and vWf levels and vWf multimeric pattern were studied in 8 patients with HSP, during active disease and twice during the remission (3 and 9 months later). Plasma vWf multimeric composition was evaluated using low resolution gels which better resolve large vWf multimers. During active disease plasma factor VIII:C, vWf:Ag, and vWf:RCoF were normal in 5% of patients and increased in three, but in each patient, platelets appeared to aggregate at doses of ristocetin lower than in normals. Furthermore, all patients demonstrated the presence of abnormally large vWf multimers usually found only in platelets and endothelial cells. Three and 9 months later, during remission, in spite of the normalization of factor VIII:C and vWf levels, the abnormal multimers were still detectable, as well as hyper-responsiveness to ristocetin. These findings confirm that damage and/or perturbation of endothelial cells is associated with HSP. Moreover, the persistence of abnormality in the vWf multimeric pattern, when the disease is inactive, suggests that the mechanisms involved operate through the entire clinical course.

The role of von Willebrand factor in haemostasis and blood loss during and after cardiopulmonary bypass surgery.

Excessive perioperative and postoperative bleeding continue to complicate cardiopulmonary bypass surgery (CPB). In this study we measured the von Willebrand factor antigen (vWF:Ag), collagen binding assay (CBA) and ristocetin cofactor assay (RiCo) in 52 patients undergoing CPB. The collagen binding assay employs the affinity of the high-molecular-weight multimers of vWF to collagen and was used in this study to demonstrate differences in the multimeric composition of vWF during and after CPB. The observed values for the vWF:Ag, CBA and RiCo were correlated to the amount of postoperative bleeding. Both the preoperative vWF:Ag and the CBA showed significant negative correlation with postoperative blood loss (r = -0.3046, P < 0.05 and r = -0.3228, P < 0.05 respectively). Higher blood loss figures correlated with lower vWF:Ag, CBA and RiCo during and after surgery with the strongest correlation 1 h post-op between both vWF:Ag and CBA and actual blood loss (r = -0.5061, P < 0.001 and r = -0.4942, P < 0.001 respectively). The strong negative correlations between vWF:Ag, CBA and RiCo before, during and after CPB and blood loss data verify the important role of vWF in primary haemostasis. Of particular note, the negative correlations between preoperative levels of vWF:Ag, CBA and postoperative blood loss provide valuable insight into another possible mechanism of excessive bleeding post CPB.

Fibrinolysis inhibits shear stress-induced platelet aggregation.

Shear stress-induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress-induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. Because these effects could mitigate shear stress-induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress-induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations > or = 2000 IU/mL, significantly inhibited shear stress-induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress-induced platelet aggregation. These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress-induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.

Plasma von Willebrand factor proteolysis in patients with chronic myeloproliferative disorders: No possibility of ex vivo degradation by calcium-dependent proteases

Patients with chronic myeloproliferative disorders(CMPD) frequently have abnormalities of plasma von Willebrand factor(vWf) multimers. The pathogenesis of this phenomenon is still unknown. In order to evaluate the possibility of ex vivo degradation of vWf during blood processing, we compared vWf antigen, ristocetin cofactor and the multimeric composition of vWf in plasmas obtained in the presence of trisodium citrate with or without calcium-dependent protease inhibitors (leupeptin, N-ethylmaleimide and Na 2EDTA). The subjects included 20 patients with CMPD, 11 with other diseases and 8 normal subjects. In patients with CMPD and normal subjects, the values of vWf antigen, ristocetin cofactor, ristocetin cofactor/vWf antigen ratio and the relative amount of large multimers of vWf did not significantly differ from each other in plasma samples with and without protease inhibitors. In other diseases, especially in a patient with disseminated intravascular coagulation, a somewhat higher amount of large multimers were found in plasma with protease inhibitors than without inhibitors. These findings indicate that the ex vivo proteolysis during blood processing is negligible in patients with CMPD, and that the observed abnormalities in vWf is an in vivo phenomenon.

Hemostatic effect of a heat-treated factor VIII concentrate (Haemate P) in von Willebrand's disease.

A heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrand's disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42-78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII: C, F VIII: Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50-75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55-76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3-4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.

Characteristics of the von willebrand factor in virus in activated F VIII concentrates: The impact of heat treatment

The known transmission of viral diseases, particularly AIDS (HIV, LAV, HTLV-III), has led to the mandatory use of virus-inactivated coagulation factor concentrates for treatment of bleeding disorders due to deficient or abnormal synthesis of the factor VIII/von Willebrand factor complex. The present investigation was undertaken to study the influence of heat-treatment on the von Willebrand factor (vWf). Using normal plasma as reference material, we studied the influence of low-purification steps in a simple cryo-plasma and a unrefined freeze-dried cryoprecipitate. For comparison, non-heated and heat-inactivated concentrates of different manufacture representing varying heat-treatment protocols were studied using quantitation of von Willebrand factor antigen (vWf:Ag) by electroimmunoassay and ELISA, and investigation of vWf multimeric composition. A locally produced factor VIII concentrate was studied before and after exposure to 68°C for 72 hours (dry state). Whenever possible, commercial preparations manufactured prior to the heat-treatment era were compared with the present product. The locally produced high purity concentrate elicited only minor changes in oligomeric satellite pattern, which did not change after dry heat exposure. In principle, no major differences were found between non-heated and pasteurized commercial concentrates of same manufactural origin.