Correction Of Bleeding Time Research Articles

Inhibition of RHOA activity preserves the survival and hemostasis function of long-term cold-stored platelets

AbstractPatients with thrombocytopenia require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions, including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis and genetic and pharmacological approaches, we identified that cold-induced activation of RAS homolog family, member A (RHOA) GTPase causes the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage–induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage–induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GPIbα. RHOA inhibition further prevents the metabolic reprogramming of cold storage–induced lesions and allows the maintenance of glycolytic flux and mitochondria-dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room temperature–stored platelets while retaining their hemostatic activity in vivo, as assessed by bleeding time correction in aspirin-treated mice. Our studies provide a mechanism-based translational approach to prevent cold storage–induced damage, which is useful for human platelet transfusion in patients with thrombocytopenia.

Antioxidant prevents clearance of hemostatically competent platelets after long-term cold storage.

Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Human Factor VIII Expression and Normalization of Bleeding Following AAV Gene Therapy in a Double Knockout Mouse Model of Hemophilia

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. Recently, Nathwani et al demonstrated in preclinical and clinical studies sustained expression of coagulation factor IX using AAV8 technology to deliver the human gene to the liver, driven by a liver specific promoter. The same group demonstrated FVIII expression in mice and primates using a modified B-domain truncated form of FVIII delivered in an AAV8 capsid.We have made an AAV5 construct containing a B-domain deleted FVIII gene (AAV5-SQ) with a liver specific promoter and evaluated it in a double knockout mouse model of hemophilia.The double knockout mice (DKO) were created by crossing factor VIII deficient mice with RAG2 deficient mice (RAG2 KO). The RAG2 KO mice lacked the ability to mount an adaptive immune response thereby allowing sustained expression of a human protein without the development of an antibody response.Eight week old male DKOs were randomly distributed into three groups, twenty per group, and treated via a single IV injection with either vehicle, or AAV5-SQ at 2 x 1013 or 1 x 1014 vg/kg. C57BL/6J mice comprised a fourth group and were treated with vehicle via a single IV injection to demonstrate wild type bleeding times and blood loss. Bleeding times and blood loss were assessed in these mice 8 weeks post-dose, at 16 weeks of age. In addition, forty 16 week old DKO mice were randomly divided into two groups and treated with a single IV injection of rhFVIII-SQ protein (rhSQ, Xyntha®) at either 50 or 200 IU/kg. Bleeding times were assessed in these mice 30 minutes post-dose, at 16 weeks of age.Eight weeks post dosing with either AAV5-SQ or vehicle, the tail bleeding time and blood loss were measured following transection of the tip of the tail for evaluation of the functional efficacy of AAV5-SQ gene therapy. Wild-type mice receiving vehicle had a mean of 0.040 ± 0.073 g blood loss and 5.11 ± 5.61 min bleeding time. DKO mice treated with vehicle had a mean blood loss and bleeding time of 0.741 ± 0.207 g and 28.96 ± 1.40 min, respectively. Mice receiving AAV5-SQ at 2x1013 vg/kg showed significantly reduced blood loss (0.387 ± 0.384 g, p=0.0008 vs DKO+ vehicle; p=0.0003 vs WT) and bleeding time (17.12 ± 11.58 min, p=0.00005 vs DKO+ vehicle; p=0.0013 vs WT) while 1x1014 vg/kg AAV5-SQ treatment corrected blood loss and bleeding times to wild-type levels (0.104 ± 0.203 g [p=0.192 vs WT, p= 5.49x10-12 vs DKO + vehicle] and 5.58 ± 9.32 mins [p=0.847 vs WT, p= 1.75x-13 vs DKO + vehicle], respectively). The effect of AAV5-SQ treatment on blood loss and bleeding time was comparable to the effects of rhSQ. DKO mice receiving 50 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.492 ± 0.297 g and 18.14 ± 9.39 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 2x1013 (p=0.343 for blood loss, p=0.760 for bleeding time). DKO mice receiving 200 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.134 ± 0.191 g and 4.29 ± 6.16 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 1x1014 (p=0.635 for blood loss, p=0.608 for bleeding time).In a separate experiment, 4 groups of DKO mice, n=10 per group, were injected with either vehicle, AAV5-SQ at 2x1013, AAV5-SQ at 2x1014 vg/kg or rhSQ at 50 IU/kg. Blood was collected 8 weeks after AAV5-SQ treatment or 5 and 30 min after rhSQ for evaluation of plasma hFVIII-SQ protein levels and activity. Expressed hFVIII-SQ levels were measured by electrochemiluminescence assay. Factor VIII-SQ protein levels at 2x1013 vg/kg AAV5-SQwere 46.8±44.0 ng/ml and 355±166ng/ml at 2x1014 vg/kg. At 50 IU/kg of rhSQ the plasma protein levels were 79.1±11.3 ng/ml at 5 min and 44.7±16.6 ng/ml at 30min post dosing. Western blot analysis of the plasma from these mice showed the expressed protein to be similar in size to rhSQ.In summary, AAV5-SQ injected into DKO hemophilic mice resulted in a dose dependent expression of B-domain deleted FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by AAV5-SQ or following exogenous administration of B-domain deleted FVIII. DisclosuresBunting:BioMarin Pharmaceutical: Employment. Zhang:BioMarin Pharmaceutical: Employment. Xie:BioMarin Pharmaceutical: Employment. Bullens:BioMarin Pharmaceutical: Employment. Mahimkar:BioMarin Pharmaceutical: Employment. Fong:BioMarin Pharmaceutical: Employment. Sandza:BioMarin Pharmaceutical: Employment. Colosi:BioMarin Pharmaceutical: Employment. Long:BioMarin Pharmaceutical: Employment. Vehar:BioMarin Pharmaceutical: Employment. Carter:BioMarin Pharmaceutical: Employment.

Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B

AbstractVon Willebrand disease (VWD)–type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

AbstractMuscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Correction of Bleeding Symptoms in von Willebrand Factor–Deficient Mice by Liver-Expressed von Willebrand Factor Mutants

von Willebrand Factor (vWF) structure-function relationship has been studied only in vitro. To investigate the physiological importance of particular vWF domains, we have introduced mutations into murine vWF (mvWF) cDNA inhibiting vWF binding to glycoprotein (Gp) Ib, GpIIbIIIa, and to fibrillar collagen. We delivered wild-type (WT) or mutant mvWF cDNA into vWF-deficient (Vwf-/-) mice using hydrodynamic injection and assessed whether hemorrhagic symptoms could be corrected. Hydrodynamic gene transfer resulted in high expression of plasma mvWF 24 hours after injection (438+/-63% for 50 microg of cDNA). Factor VIII activity was normalized in Vwf-/- mice injected with mvWF cDNA and multimerization was achieved. Bleeding time was corrected after injection of WT mvWF cDNA in Vwf-/- mice whereas noninjected mice did not stop bleeding. Injection of the GpIIbIIIa and the collagen binding mutants in Vwf-/- mice also resulted in a correction of bleeding time whereas mice injected with the GpIb binding mutant were bleeding for as long they were observed, although blood loss was decreased compared with noninjected mice (61+/-21 microL versus 232+/-63 microL). Our model allows the rapid in vivo evaluation of specific mutations on plasma vWF function.

Treatment of von Willebrand disease with a high-purity factor VIII/von Willebrand factor concentrate: a prospective, multicenter study: Presented in part at the meeting of the International Society of Thrombosis and Haemostasis in Washington, DC, August 1999.

AbstractAmong patients with von Willebrand disease (VWD) who are unresponsive to desmopressin therapy, replacement with plasma-derived concentrates is the treatment of choice. Because prospective studies are lacking, such treatment has been largely empirical. A multicenter, prospective study has been conducted in 81 patients with VWD (15 patients with type 1, 34 with type 2, and 32 with type 3 disease) to investigate the efficacy of a high-purity factor VIII/von Willebrand factor (FVIII/VWF) concentrate for treatment of bleeding and surgical prophylaxis. Two preparations of the concentrate—one virally inactivated with solvent detergent, the other with an additional heat-treatment step—were evaluated. Pharmacokinetic parameters were similar for both preparations. Using pre-established dosages based on the results of pharmacokinetic studies, 53 patients were administered either preparation for the treatment of 87 bleeding episodes, and 39 patients were treated prophylactically for 71 surgical or invasive procedures. Sixty-five (74.7%) and 10 (11.5%) of the bleeding episodes were controlled with 1 or 2 infusions, respectively. Patients with severe type 3 VWD typically required more infusions and higher doses, at shorter time intervals, than did patients with generally milder types 1 and 2. Among patients undergoing surgical procedures, blood loss was lower than that predicted prospectively, and losses exceeding the predicted value did not correlate with the postinfusion skin bleeding time. In conclusion, the concentrate effectively stopped active bleeding and provided adequate hemostasis for surgical or invasive procedures, even in the absence of bleeding time correction.

Hemostasis in patients with severe von Willebrand disease improves after normal platelet transfusion and normalizes with further correction of the plasma defect.

A defective hemostatic effect of plasma concentrate infusion in patients with severe von Willebrand disease (vWD) has been ascribed to the absence of platelet von Willebrand factor (vWF) STUDY DESIGN AND METHODS: The role of platelet vWF in hemostasis of severe vWD was investigated. A plateletpheresis unit (4-5 x 10(11) platelets) from a normal compatible donor was transfused before any cryoprecipitate infusion to three type 3 vWD patients and to one patient with severe type 1 vWD with low levels of platelet vWF who required replacement therapy for bleeding episodes. Autologous platelets were transfused to one of the patients with type 3 vWD. Partial corrections of bleeding times (14-17 min vs. baseline >30 min) were observed in all patients after the transfusion of normal platelets. During cryoprecipitate infusion, bleeding times were normalized (<6 min), and bleeding episodes stopped when plasma levels of vWF activity ranged from 14 to 18 U per dL. Platelet interactions with the subendothelium increased in parallel with the correction of bleeding times. These results indicate that if approximately 20 percent of the total number of platelets have normal vWF antigen and if plasma vWF levels are at least 14 U per dL, then bleeding times will normalize and mucosal hemorrhages will stop. Transfusion of autologous platelets in one patient with type 3 vWD did not modify bleeding times or platelet adhesion on the subendothelium. The hemostatic effect of normal platelets in type 3 vWD seems to be related to the platelet vWF in the transfused platelets.

1-Deamino-8 D-arginine vasopressin decreases the production of 13-hydroxyoctadecadienoic acid by endothelial cells

1-Deamino-8 D-arginine vasopressin (DDAVP) has been used effectively to normalize the bleeding time in various hemostatic disorders. In von Willebrand disease the reduction in bleeding time is due to the preferential release of large multimers of von Willebrand factor from endothelial cells. However, since the bleeding time correction in patients with uremia and liver disease is independent of the release of von Willebrand antigen and activity, other mechanisms of action of DDAVP need to be considered. Endothelial cells generate several thromborepellant factors including 13-hydroxyoctadecadienoic acid (13-HODE), an inhibitor of platelet adhesion to subendothelium. Using cultured fetal bovine aortic endothelial cells (FBAECs), we have investigated whether DDAVP modulates the production of 13-HODE. We have demonstrated that 14C-linoleic acid labeled FBAECs release several oxygenated derivatives of linoleic acid following a 120 min incubation in the presence of serum. One of these products was identified by chromatographic procedures as 13-HODE. The production of 13-HODE was decreased significantly by DDAVP (1–100 ng/ml) with maximal reduction (approx. 25%) seen at 1 ng/ml of DDAVP. While vehicle treated control FBAECs generated 6780 ± 690 cpm of 13-HODE per 10 6 cells (mean ± SE, n=8), DDAVP treated FBAECs produced 4950 ± 310 (P < 0.01), 5390 ± 390 (P < 0.01), and 5720 ± 410 cpm (P < 0.05) of 13-HODE at 1, 10, and 100 ng/ml DDAVP respectively. Our findings of a decrease in 13-HODE would explain the previously observed morphologic changes of increased platelet adhesion to subendothelium following DDAVP infusion and contributes to our understanding of the mode of action of this therapeutic agent in hemostatic disorders.

Clinical indications for desmopressin (DDAVP) in congenital and acquired von Willebrand disease

In the majority of patients with congenital and acquired von Willebrand disease (vWD), desmopressin (DDAVP) is able to increase circulating factor VIII coagulant (VIII:C) to levels sufficient to secure satisfactory hemostasis. The bleeding time (BT) is also often normalized. In this review, all cases of vWD treated with DDAVP for the prevention or control of hemorrhage and reported in the literature for whom at least basal and peak values of VIII:C were available have been analysed. When reported, the effect on the BT was also considered. It appears that, in keeping with clinical experience gained with blood products, the correction of VIII:C defect is often sufficient to secure normal hemostasis. The only significant exception is mucosal bleeding, for which the correction of BT also appears to be necessary. Several patients (mainly with type I vWD) with basal VIII:C levels of 5–10% have been successfully treated to prevent bleeding after tooth extractions and minor surgery and to control spontaneous and post-traumatic bleeding. Experience with DDAVP in major surgery is still limited, so that the compound cannot be recommended for routine use. In acquired vWD, a trial with DDAVP is advised before resorting to substitutive therapy with blood derivatives. Since side effects to DDAVP treatment are limited and no major complications have been consistently demonstrated, DDAVP can be proposed as the treatment of first choice for most patients with vWD. The recent availability of concentrated preparations of DDAVP for intranasal administration and the demonstration that the subcutaneous route is an effective and simpler alternative to the intravenous route should further facilitate its use and make home-therapy feasible.

Hermansky-Pudlak syndrome: correction of bleeding time by 1-desamino-8D-arginine vasopressin.

The effect of the synthetic vasopressin derivative 1-desamino-8D-arginine vasopressin (DDAVP = desmopressin) on bleeding time was studied in three patients with Hermansky Pudlak syndrome. A good response was observed in this type of storage pool disease. DDAVP might be useful in managing the bleeding disorder found in patients with the Hermansky-Pudlak syndrome.

The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.

The Heterogeneity of Type IIA von Willebrand’s Disease: Studies With Protease Inhibitors

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8-D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.

Continuous-flow techniques for platelet concentrate collection: a step toward standardization and yield predictability.

This study chronicles leukocyte- and erythrocyte-depleted platelet concentrate collection by a dual stage channel in which three variables: 1) donor peripheral blood platelet concentration, 2) total blood processed, and 3) collection volume were statistically correlated with platelet yield as determined by a multiple regression analysis of single variables. Platelet concentration in the final yield was related to donor precount and collection rate, and could be varied as indicated for individual applications. Total blood processed was established by procedure time, which in turn was defined by citrate-induced calcium changes in the donor. Reduction in peripheral blood platelet concentration averaged 24% for a mean platelet yield of 3.8 X 10(11). An average of 40% of transfused platelet concentrates were recovered in recipient peripheral blood 1 hr posttransfusion and were hemostatically effective, as determined by correction of bleeding time. Platelet yields and patient response were sustained during current collection procedures, verifying the principles described during the investigative period.

Automated platelet production during plasmapheresis.

Manual plasmapheresis is widely used to permit collection of fresh-frozen plasma with return of the red cells to the donor. However, this method is time-consuming and carries the inherent risk of returning the wrong cells to any individual donor. Automated plasmapheresis, using a specially designed discontinuous cell separator, permits the collection of 500 ml of plasma within 30 minutes without disconnecting the donor from the bag containing the donor's red cells. However, this plasma contains a small number of platelets which are not suitable for transfusion. We now report the modification of this machine to permit simultaneous collection of 3 units of platelet concentrate as well as 500 ml of plasma in less than 50 minutes. Reduction of the g force of the separator from 1200 to 650 g permits the simultaneous collection of plasma and a platelet concentrate with an average yield of 2.21 X 10(11) platelets. Platelet size distribution, in-vitro function. 51Cr survival, posttransfusion increment, and bleeding time correction are all normal. This modification has increased the flexibility of the separator and provides an instrument which can be used with a random volunteer blood donor population to generate both plasma and cells in less than 50 minutes and with increased cost-effectiveness over plasmapheresis alone.

Experience with D.D.A.V.P. in the correction of bleeding time in patients with von willebrand’s disease

1-Deamino-8-D-arginine vasopressin (D.D.A.V.P.), a synthetic analogue of anti-diuretic hormone, when administered intravenously produces a dose related rise in Factor VIII coagulant activity (VIII:C), Factor VIII related antigen (VIII R:AG) and VIII Ristocetin Cofactor (VIII:Rcof) in patients with mild haemophilia and von Willebrand’s disease. The published studies have shown that D.D.A.V.P. in the dose range .2-.5 μg/kg produces little or no shortening in bleeding time. Eight patients with classical von Willebrand’s disease were studied. Following intravenous administration of D.D.A.V.P. in a dose range .3-.6 μg/kg body weight, a shortening of bleeding time was observed in all cases. Six of these patients were studied in an attempt to demonstrate a correlation between the shortening of the bleeding time and rise in Factor VIII:C, Factor VIII antigen, and VIII Rcof. This reduction in bleeding time, which lasted 3 to 5 h in the 6 patients who were serially studied, is related to increases in all factors measured, although only Factor VIII:C consistently reached normal levels. Despite the correction of bleeding time, the Factor VIII Rcof activity did not approach the normal range in any of the subjects, and was barely detected in one patient who had a normal bleeding time after D.D.A.V.P. infusion. The pattern of Factor VIII:Fcof response was similar in members of the same family. These results suggest that Factor VIII: Rcof activity and the factor responsible for correction of bleeding time are separate entities.