Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome.

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

A reassessment of the bleeding time: association of age, hematocrit, platelet function, von Willebrand factor, and bleeding time thromboxane B2 with the length of the bleeding time.

In order to provide an overview of the relative contribution of platelet, von Willebrand factor, and other abnormalities to patients with clinical bleeding difficulties, we performed a retrospective survey of coagulation studies on 569 individuals referred to the University of Manitoba coagulation laboratory because they, or a closely related family member, showed clinical evidence of a bleeding disorder. There was a highly significant (p less than 0.001) negative correlation between the bleeding time and each of the following parameters: the platelet count; the hematocrit; the percent aggregation to collagen, epinephrine, ADP, and arachidonic acid; and the logarithm of von Willebrand factor antigen and a measure of its activity (ristocetin cofactor). A significant and independent inverse relationship between the length of the bleeding time and the extent of platelet adhesion to glass beads, patient age, and prothrombin consumption were also observed. Multivariate analysis of the ability of all parameters to predict the bleeding time showed an r2 of only 0.33. Bleeding time thromboxane B2, in a second smaller study of 70 patients, showed a negative correlation with the length of the bleeding time (p = 0.0001), and, when used together with the above parameters, significantly enhanced the ability to predict the length of the bleeding time (r2 = 0.55). Defects in platelet function, as measured in vitro, and significant enough to have an effect on the bleeding time, occurred with greater frequency than defects in von Willebrand factor in the Manitoba patients evaluated.

Relationship between human development and disappearance of unusually large von Willebrand factor multimers from plasma.

von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF. There was no significant relationship between either gestational age at time of delivery or birth weight and likelihood of finding ULvWF multimers in cord PPP samples. No maternal PPP sample contained ULvWF multimers. Serial heelstick samples from 16 preterm and term neonates were analyzed for 8 weeks. ULvWF multimers disappeared from the PPP of ten of the neonates during this time. The PPP of four neonates had vWF patterns similar to those in normal adult PPP throughout the sampling period. The ULvWF multimeric forms of fetal and neonatal PPP samples were similar to those constitutively released from endothelial cells. They were not as slowly migrating in a very porous 0.5% agarose gel system as the ULvWF multimers released from Weibel-Palade bodies in response to the calcium ionophore A23187. A vWF protomer was present in 97% of fetal samples, 83% of cord blood specimens, and 11% of neonatal heelstick samples, but was not found in any maternal sample. These results indicate that control mechanisms operative in older children and adults to prevent circulation of ULvWF multimers and vWF protomeric forms are normally acquired late in uterine life or during the neonatal period. ULvWF multimers, which are normal components of fetal, most cord, and some neonatal plasma samples, may contribute to in utero and postnatal hemostasis.

Clinical effectiveness of desmopressin in a case of acquired von Willebrand's syndrome associated with benign monoclonal gammopathy.

A case of acquired von Willebrand's syndrome (avWs) secondary to benign monoclonal gammopathy, is described, in which desmopressin (DDAVP) has proven effective repeatedly in preventing bleeding after tooth extraction. The laboratory pattern was similar to that of congenital type IA von Willebrand's disease. After DDAVP, prolonged bleeding time and factor VIII/von Willebrand factor activities were normalized. The disappearance rate of the elicited activities was similar to that observed in patients with congenital disease. This report adds to the scarce data concerning the haemostatic effectiveness of DDAVP in avWs and suggests that this agent might also be used in controlling or preventing bleeding in patients with the acquired disease, selected on the basis of their biological responsiveness to a test-infusion.

Features of endothelial dysfunction in early diabetic nephropathy.

The release of tissue plasminogen activator (tPA) by vascular endothelial cells during exercise was studied in forty men with insulin-dependent diabetes. Three groups, matched for age and diabetes duration, were defined as: group I (n = 19), normal urinary albumin excretion (less than 30 mg/24 h); group II (n = 11), incipient diabetic nephropathy (30-300 mg albumin excreted per 24 h); and group III (n = 10), clinical diabetic nephropathy (more than 300 mg albumin excreted per 24 h). Nine non-diabetic men served as controls. The rise in tPA antigen with exercise was similar in the controls and group I but significantly smaller in groups II and III (p less than 0.01). The albumin transcapillary escape rate was significantly higher in groups II and III than in group I and normal controls (p less than 0.01). The basal plasma level of von Willebrand factor was higher in groups III (p less than 0.01) and II (difference not significant, p = 0.06) than in group I and normal controls. These findings suggest that insulin-dependent diabetic patients with only slightly raised urinary albumin excretion have general endothelial cell dysfunction or damage. It is not yet clear whether these changes are important in the pathogenesis of thrombosis and atherosclerosis in these patients.

Changes in von Willebrand factor during cardiac surgery: effect of desmopressin acetate.

Patients who receive desmopressin acetate (dDAVP) after cardiopulmonary bypass bleed less during operation and in the first 24 hours after operation than do patients who receive a placebo. To study the mechanism of improved hemostasis in bypass patients, we examined the relationship between von Willebrand factor (vWF) and blood loss in 70 cardiopulmonary bypass patients, one-half of whom received desmopressin intraoperatively. vWF concentration and multimeric composition were analyzed before and after bypass, after drug treatment, and 24 hours after operation. Before operation, patients with valvular disease had lower percentages of vWF high-mol-wt multimers (HMWMs) than did healthy subjects or patients with coronary artery disease, but subsequent blood loss, vWF activity, and bleeding times were not related to this finding. Irrespective of drug treatment, patients who had low preoperative vWF and who had a net loss of the protein during bypass bled more after bypass than did similar patients who had a net increase of vWF during bypass. HMWMs rose to above normal levels after bypass regardless of desmopressin infusion. Differences in the concentration of vWF between desmopressin and placebo patients after receipt of the drug, although small, were better correlated with reduced blood loss than were differences in HMWM distribution. We conclude that the beneficial effect of desmopressin on hemostasis following cardiopulmonary bypass cannot be attributed to a drug-induced change in HMWM distribution but may be related to an increase in overall vWF concentration.

Asialo-von Willebrand factor inhibits platelet adherence to human arterial subendothelium: discrepancy between ristocetin cofactor activity and primary hemostatic function.

Platelet adherence at high wall shear rates requires plasma von Willebrand factor (vWF). Clinically, the ristocetin cofactor (RCof) activity is the only widely available assay for vWF function. When purified vWF is treated with neuraminidase to yield asialo-vWF (AS-vWF), its RCof activity is increased by 20% to 40%. AS-vWF binds to normal human platelets independently of ristocetin and induces platelet aggregation in the presence of fibrinogen. To determine whether AS-vWF also shows an enhanced capacity to support platelet adherence to subendothelium, we used the Baumgartner technique. Intact vWF, AS-vWF, or AS-vWF treated with beta-galactosidase (asialo, agalacto-vWF; AS,AG-vWF) was added to normal citrated whole blood before perfusion over human umbilical artery segments (wall shear rate, 2,600 sec-1). Four micrograms per milliliter AS-vWF caused a 69% reduction in total platelet adherence compared with citrated whole blood (P less than .001), and 4 micrograms/mL AS,AG-vWF led to a 48% reduction (P less than .005). With 4 micrograms/mL intact vWF, the platelet adherence values were not significantly different from the controls. No significant differences in subendothelial platelet thrombi or postperfusion platelet counts were evident among any of the groups. In reconstituted afibrinogenemic perfusates, 4 micrograms/mL AS-vWF caused a 42% reduction in platelet adherence (P less than .05). Thus, AS-vWF is a potent inhibitor of platelet adherence, despite its enhanced RCof specific activity. Abnormalities in vWF carbohydrate may play a role in impaired primary hemostasis in some patients with von Willebrand's disease.

Distribution of von Willebrand factor in porcine intima varies with blood vessel type and location.

The von Willebrand factor (vWF) has been generally accepted as a marker for endothelial cells. In a systematic immunolocalization study of porcine blood vessels that used indirect immunofluorescence with a monospecific polyclonal anti-vWF and two monoclonal anti-vWFs, we observed that vWF is not universally distributed in intact, fresh endothelia. vWF is consistently localized in veins, with the exception of the pulmonic vein. In arteries, vWF is generally absent except for areas of the distal abdominal aorta, the vaso vasorum of the thoracic aorta, and the pulmonic artery. We conclude that there are regional differences in the distribution of vWF in the various endothelial beds of pigs.

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome.

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

The von Willebrand factor in myocardial infarction and unstable angina: a kinetic study.

Recent studies have demonstrated elevations of von Willebrand Factor following acute myocardial infarction (AMI). In order to determine if this parameter may serve as a marker for AMI, we tested the blood levels of vWF and Factor VIII:C in 28 patients with AMI, 9 patients with unstable angina, 7 patients with atypical chest pain, and 25 healthy volunteers. The level of ristocetin cofactor activity of vWF was between 70 and 144% in the control group. In patients with AMI, the mean level of this activity was 175% on the first day following infarction, rose to a peak of 270% on the fifth and sixth days, and was still significantly greater than normal in all patients on the 14th day. The vWF:Ag level closely paralleled the rise of ristocetin cofactor activity of vWF, with a peak of 336% on day 5. FVIII:C was not significantly changed. No significant elevation of vWF was observed in patients with unstable angina. The ristocetin cofactor activity of vWF and vWF:Ag thus are sensitive biochemical indicators for recent AMI, and may serve as useful markers for up to 14 days following infarction, when the traditional enzymes have returned to normal levels.

Effects of fresh-frozen plasma and its cryosupernatant fraction on von Willebrand factor multimeric forms in chronic relapsing thrombotic thrombocytopenic purpura.

Remission plasma samples of some patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP) contain unusually large von Willebrand factor (vWF) multimers similar to those produced by normal human endothelial cells in culture. The infusion of the cryosupernatant fraction of normal plasma is as effective as normal fresh-frozen plasma (FFP) in the treatment or prevention of TTP episodes in patients with the chronic relapsing form of TTP. Three patients with chronic relapsing TTP during remission have unusually large vWF multimers present in their plasma. Two of the patients were transfused once with FFP, one of the two received cryosupernatant on three occasions, and the third patient was studied before and immediately after plasma exchange. Unusually large vWF multimers decreased or disappeared from patient plasma samples within 1/2 to 1 1/2 hours following the transfusion of FFP (on two occasions) or cryosupernatant (on two of three occasions), and immediately after plasma exchange (on one occasion). The patient who received cryosupernatant was studied serially after the infusions. Unusually large vWF multimers returned to her plasma within ten to 24 hours and persisted thereafter. Unusually large vWF multimers did not disappear from patient remission plasma samples, or from the culture medium removed from normal human endothelial cells, when these fluids were incubated in vitro with either normal FFP or cryosupernatant. We conclude that an activity in FFP, and its cryosupernatant fraction, promoted the rapid in vivo disappearance of unusually large vWF multimers from the plasma of two patients with chronic relapsing TTP in remission, and plasma exchange reversed the abnormality in a third patient who was in partial remission. Neither FFP nor cryosupernatant directly converted unusually large multimers to smaller vWF forms in vitro in the fluid phase. These results indicate that an activity in the cryosupernatant fraction of normal plasma is involved in vivo in controlling the metabolism of unusually large vWF multimers, and that this process is defective in some chronic relapsing TTP patients.

Destruction of ristocetin cofactor by coagulation at 4 degrees C.

Ristocetin cofactor (VIIIR:RCo) and factor VIII-related antigen (VIIIR:Ag) were measured in anticoagulated and non-anticoagulated blood incubated at 4 degrees C, room temperature (RT) or 37 degrees C for 24 hours. A marked decrease in VIIIR:RCo, to almost undetectable levels, and a smaller decrease in VIIIR:Ag occurred when whole blood clotted at 4 degrees C. These changes were slight or absent when blood clotted at RT or 37 degrees C. VIIIR:RCo lost at 4 degrees C was not recoverable by further incubation at 37 degrees C but the less-marked loss of VIIIR:Ag was partially recovered. In blood which had clotted at 4 degrees C there was a change in the electrophoretic profile of VIIIR:Ag on crossed immunoelectrophoresis: there was more anodal migration of the VIIIR:Ag peak, consistent with a decrease in the mean molecular size. Further experiments showed that the decrease in VIIIR:RCo during coagulation at 4 degrees C preceded the decrease in fibrinogen levels. In cell-free plasma VIIIR:RCo also decreased markedly when coagulation occurred at 4 degrees C. The results show that loss of VIIIR:RCo occurs when blood is allowed to clot at 4 degrees C: this is not due to cryoprecipitation and does not require the presence of blood cells. The data suggest that it is probably caused by plasma proteases activated early in the coagulation pathway.

Factor VIII activity and thyroid function.

Plasma factor VIII coagulant activity is decreased in hypothyroid patients and increased in hyperthyroid patients. We studied 21 untreated hypothyroid patients. Factor VIII coagulant activity was mildly decreased in association with significant depression of factor-VIII-related antigen and ristocetin cofactor activity in five patients. Factor-VIII-related properties significantly increased with oral thyroid replacement therapy in seven of 10 patients. Twenty-two untreated hyperthyroid patients were similarly evaluated. In 21 of these patients significant increases were noted in factor VIII coagulant activity, factor-VIII-related antigens, and ristocetin cofactor activity. Elevated factor-VIII-related properties returned to normal in all of 10 patients treated with radioactive iodine or propylthiouracil. We discuss the relation between thyroid function and factor-VIII-related properties in both hypothyroid and hyperthyroid patients.