Among inherited risk factors for venous thrombosis, the most common are the FV-G1691A and FII-G20210A polymorphisms. The FV-G1691A polymorphism is preferentially observed in Europe, with differences between European countries. The FII-G20210A polymorphism is observed all over the world. The study was designed to compare the prevalence of the FV-G1691A and FII-G20210A polymorphisms in a large French population of unrelated individuals with no thrombotic disease history and to determine the age and geographical distributions. Over a period of 18 months, 6154 individuals were included throughout France and FV-G1691A and FII-G20210A polymorphisms were determined. The FV-G1691A prevalence was 3.84% (95% confidence interval 3.35–4.33) and the FII-G20210A prevalence was 3.07% (95% CI 2.63–3.51). A north-east/south-west gradient was observed in the FV-G1691A geographical distribution. No difference was observed in the geographical distribution of FII-G20210A polymorphism nor in the age distribution of the two polymorphisms. The prevalence of the two polymorphisms was similar whatever the blood group (O or non-O). Plasma D-dimers were significantly higher in healthy individuals with FV-G1691A but not in individuals with FII-G20210A. Thirty percent of variation in plasma prothrombin level was explained by environmental factors (serum cholesterol, age, oral contraception, hormonal replacement therapy, body mass index, sex) and genetic factors (FII-G20210A). As expected, individuals with FII-G20210A displayed higher plasma prothrombin level compared with individuals with wild type. However, this was not associated with a modification of the fibrin clot elastic modulus. This study shows a differential distribution of the two polymorphisms among the French territory. These polymorphisms confer a very mild hypercoagulable state as shown by the limited increased in basal D-dimers in mutated FV-G1691A populations and only a trend that does not reach statistical significance for FII-G20210A population.

Replacement therapy is the treatment of choice for patients with von Willebrand disease who are unresponsive to desmopressin. In order to prevent transmission of non-enveloped viruses, a solvent/detergent-treated plasma-derived von Willebrand factor available in France since 1989 has been subjected to additional removal/inactivation steps by 35 nm filtration and dry heating for 72 h at 80 degrees C. This preclinical study evaluates the potential immunogenicity of this new product by comparing the antibodies raised in pigs affected with von Willebrand disease after intravenous injection of either a solvent/detergent-treated product or a triple-secured product. Our data showed that there is no difference between the two products in terms of the rate and intensity of the humoral response measured by both binding and neutralizing antibody levels. It was concluded that no antigenic alterations of von Willebrand factor molecules during the nanofiltration and final dry-heating steps were detected in our animal model. (c) 2005 Lippincott Williams & Wilkins.

Background. The clinical development of liver-support devices based on perfusion of either pig hepatocytes cartridges or whole pig livers has been hampered by the ability to use sufficient liver cell mass to provide adequate metabolic support, limited perfusion times, and the potential for patient exposure to pig zoonotic diseases.Methods. We designed an original system in which an isolated intact pig liver was perfused extracorporeally under physiological conditions in a closed loop circuit with allogeneic pig blood and constant monitoring of major physiological and functional parameters. The perfusion circuit further included an interface membrane to provide for separation of patient and liver perfusion circulation.Results. Prolonged (6-21 hr) liver perfusion did not produce significant liver damage as reflected by modest rises in the levels of the serum transaminases, stability of main biochemical parameters (including potassium), and the maintenance of normal cellular morphology. Optimal liver function was documented as measured by lactate consumption, control of glycemia, and the results of clotting studies and functional assays. The perfused liver cleared 82% and 79% of peak bilirubin and ammonia concentrations with clearing kinetics identical throughout perfusion. Indocyanine green clearance was identical to that observed in the living donor before explant surgery.Conclusions. In conclusion, the extracorporeal pig liver perfusion apparatus described here allows optimal pig liver function for prolonged periods of time. The microporous membrane to provide separation of donor organ and recipient and the high level of functional activity suggest that this form of liver metabolic support may have important clinical applications.

To evaluate the regulation of plasma von Willebrand factor (vWF) and its in situ production by endothelial cells (ECs), 12 swine leukocyte antigen (SLA)-compatible left lung transplantations were performed. Normal lungs were transplanted into 10 pigs homozygous for von Willebrand disease and into 2 normal pigs. Additionally, 1 normal pig underwent pneumonectomy, and 1 SLA-incompatible lung transplantation between normal pigs was performed. None of the transplanted animals received immunosuppressive therapy. Plasma vWF level was evaluated by ELISA and multimeric pattern. EC vWF content was assessed by immunohistochemistry. Global hemostasis was assessed by standardized ear bleeding time. Six of 12 SLA-compatible lung transplantations and the incompatible transplantation were successful and were used for the study. The functions and the viability of ECs, reflected by their ability to produce vWF and normal multimeric plasma vWF pattern, were preserved in SLA-compatible and -incompatible lung transplantations. vWF production was preserved in ECs that initially synthesized it. EC constitutive and storage pathways are modulated differently according to transplantation compatibility and severity of rejection. In SLA-compatible lung transplantations without histological evidence of rejection, the production of vWF was preserved, whereas constitutive vWF secretion appeared to be altered in cases with minor histological signs of rejection. In pigs with von Willebrand disease that were transplanted with normal lungs without sign of rejection, plasma vWF was significantly increased in an amount expected from the estimated production of a normal lung. In the transplanted normal lung, there was no vWF overexpression by the ECs and no recruitment of ECs that initially did not express vWF. In SLA-incompatible transplantation, ECs were morphologically normal with increased and blurred vWF labeling, whereas plasma vWF levels remained normal, reflecting that EC activation is associated with an increased vWF production with probable diversion to storage pathway. This model depicts the changes of EC regulation of vWF secretion in pig lung transplants. However, this model cannot be directly extrapolated to human organ transplantation because animals did not receive any immunosuppressive therapy, which may be toxic to ECs.

Les porcs atteints de maladie de Willebrand (vWD) homozygote, affection hemorragique autosomale, developpent une maladie severe identique a la vWD humaine de type 3; les animaux homozygotes Willebrand sont caracterises par un allongement du temps de saignement (TS > 30 minutes), un taux inferieur aux valeurs de detection de l'antigene du facteur Willebrand (vWF) et de son activite, une faible activite du facteur VIII (FVIII), ex vivo une importante diminution de l'adhesion plaquettaire et de la thrombogenese a 1 600 s -1 , et in vivo une absence de reponse thrombotique consecutive a une lesion et une stenose vasculaire. Le porc vWD a permis des avancees dans la comprehension du role et des fonctions du vWF dans l'hemostase, la thrombose et l'atherosclerose, en particulier par la creation de greffes croisees entre animaux normaux et vWD (greffes de moelle osseuse, greffes de poumons, organes riches en cellules endotheliales, transi fert de cellules endotheliales). Il a aussi ete utilise pour evaluer plusieurs produits de substitution (concentres de vWF de differentes origines). L'ensemble des etudes realisees avec du materiel autologue ou allogenique permet d'evaluer des protocoles qui pourraient etre menes chez l'homme. Les etudes conduites avec du materiel xenogenique (en particulier pour tester des concentres therapeutiques derives du plasma humain ou recombinants) se heurtent a une difficulte: il existe des differences moleculaires entre les molecules de vWF humain et porcin, minimes mais qui entrainent une immunogenicite des produits humains perfuses au porc. En prenant en compte ces differences, le porc Willebrand est un outil utile pour la conception et l'evaluation de therapeutiques substitutives innovantes.

Deep vein thrombosis (DVT) is a common complication of paraplegia despite prophylactic anticoagulant therapy. The diagnosis relies primarily on ultrasonography or phlebography; these investigations are difficult, expensive and can be time-consuming in paraplegic patients. Study Design: To evaluate the usefulness of coagulation activation markers in excluding a diagnosis of DVT, D-Dimers, thrombin-antithrombin complexes, prothrombin fragments (F1+2) and activated factor VIIa. Objectives: To improve the diagnosis of deep venous thrombosis in paraplegic patients. Setting: This collaborative work was done at Raymond Poincaré Hospital, Garches, France. Methods: To evaluate the usefulness of coagulation activation markers in excluding a diagnosis of DVT, D-Dimers (D-Di), thrombin-antithrombin (TAT) complexes, prothrombin fragments (F1+2) and activated factor VIIa (FVIIa), were determined in a prospective study of 67 consecutive patients with paraplegia or tetraplegia. Doppler ultrasonography and/or phlebography of the lower limbs and D-Di, TAT, F1+2 level determination were systematically done in each patient at admission to our rehabilitation unit. Results: Despite prophylactic low molecular weight heparin therapy, six of the 67 patients developed DVT diagnosed by radiologic explorations. D-Di levels measured by a reference ELISA (Asserachrom D-Di, Diagnostica Stago) or a new rapid automated turbidimetric test (STA-Liatest D-Di) were greater than 500 ng/ml in all DVT patients and in 40 non-DVT patients, of whom most had urinary tract infections, osteomas, or pressure sores. D-Di values were normal in only 21/67 patients (31%). The negative predictive value of D-Di in our study was 100% since all DVT patients had D-Di values greater than 500 ng/ml. TAT and F1+2 levels were not correlated with D-Di levels but also had a negative predictive value of 100%. Comparison of D-Di levels obtained using the two tests showed that results of the reference ELISA were closely correlated to those of the new rapid automated turbidimetric. TAT, F1+2, and factor VIIa are not useful for measuring hypercoagulability in paraplegic or tetraplegic patients since no rapid tests for determining these parameters are available. Conclusion: D-Di levels determined using an ELISA or a new rapid automated turbidimetric test have a good negative predictive value for DVT in paraplegic or tetraplegic patients and may reduce the need for Doppler ultrasonography and/or phlebography by 31%.

Thienopyridines (ticlopidine or clopidogrel) alone or in combination with aspirin are now the reference antiplatelet therapy after stent implantation. To better understand the high efficacy and low risk of bleeding with these agents, we tested clopidogrel alone or with aspirin in an acute ex vivo flow chamber model and in a subacute in vivo arterial thrombosis model. Clopidogrel induced a dose-dependent increase in bleeding time (BT), inhibited ADP-induced platelet aggregation and in the flow chamber reduced thrombus size, and changed thrombus structure to broad-based structure composed of nondegranulated loosely attached platelets contrasting with the tight clumps of degranulated platelets seen without clopidogrel. The in vivo model involved angioplasty and stenting at the site of a preinduced arterial lesion and thrombosis in pig carotid arteries. Clopidogrel alone or with aspirin (but not aspirin alone) decreased the number of stented vessels occluded for more than 24 h and conversely reduced the number of occluding thrombus. At 96 h after stenting, 100% and 90% of the arteries were patent with clopidogrel/aspirin and clopidogrel alone, respectively (vs. 67% and 44% with aspirin and saline, respectively). Clopidogrel destabilizes thrombus without complete abolishment of platelet reactivity.

BACKGROUND:The coagulation process in hyperacute and delayed xenograft rejection is essential and depends upon platelet adhesion and aggregation. The initial binding of platelets to the damaged endothelium is due to the interaction of the platelet receptor glycoprotein Ib with von Willebrand factor (vWF), which is present on activated endothelial cells and bound to the subendothelial matrix. We hypothesized that the use of organs from animals with homozygous von Willebrand disease (vWD), severely deficient in vWF, might prevent the thrombosis encountered in delayed xenograft rejection.METHODS:Ten baboons were treated by extracorporeal immunoadsorption of xenoreactive natural antibodies (XNA) through the donor pig liver to inhibit hyperacute rejection and received heterotopic vWD or control pig kidney xenografts. XNA levels, coagulation, and platelet activation markers were studied, and specimens of rejected kidneys were analyzed histologically.RESULTS:Although XNA depletion was comparable in both groups, neither kidney function nor survival times of control (n=5) or vWD (n=5) porcine kidneys showed any difference. Platelet and coagulation activation was evidenced in both groups after surgery and at rejection time. Immunohistochemical analysis revealed a weak endothelial vWF immunostaining in the rejected vWD kidneys, whereas it was undetectable in the nongrafted vWD kidneys, suggesting the deposition of baboon plasma vWF on the porcine vessels.CONCLUSIONS:The use of vWD organs did not improve the survival time of grafted kidneys in this xenotransplantation model. Further studies on the use of vWD organs, in association with other therapeutic approaches, such as complement inhibition, are nevertheless necessary to evaluate the usefulness of vWF deficiency as an adjunctive therapy to decrease the coagulation process during xenograft rejection.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

To evaluate the relative role of plasma and platelet von Willebrand factor (vWF) pools in hemostasis and arterial thrombogenesis, pigs with vW disease (vWD) were injected with vWF concentrate and/or grafted with bone marrow from a normal pig. Hemostasis was assessed by measurement of ear immersion bleeding time, factor VIII (FVIII) activity, and plasma and platelet vWF antigen levels. The thrombotic process was explored at 650 s(-1) and 1600 s(-1) in an ex vivo cylindrical perfusion chamber. Pigs with vWD exhibited a prolonged bleeding time (>30 minutes) compared with normal pigs (<5 minutes); in addition, they showed normal platelet adhesion and thrombus formation at 650 s(-1) but profoundly reduced platelet adhesion and thrombus formation at 1600 s(-1). Each experiment was performed before and 3 and 24 hours after injection of vWF concentrate. In our bleeding time study, only plasma vWF restoration induced a partial but delayed correction (24 hours postinjection), which was correlated with the highest measured level of FVIII activity. In the perfusion chamber model, restoration of plasma or platelet vWF pools resulted in similar partial correction of platelet adhesion and average thrombus size. In the perfused pigs, the maximum correction occurred 3 hours postinjection. Platelet deposition reached normal values after vWF concentrate was injected into a grafted pig. The present results suggest that when both plasma and platelet vWF levels are restored in vWD pigs, bleeding time and the thrombotic process are normalized according to different kinetics and with differing degrees of effectiveness.

OBJECTIVE To establish the feasibility and safety of recuperating blood absorbed by swabs used during orthopaedic surgery. STUDY DESIGN Open, prospective study. PATIENTS Included were children undergoing potentially haemorrhagic orthopaedic surgery for whom intraoperative blood salvage seemed possible. Excluded were those with contraindications for this procedure such as septic surgery and cancer surgery. METHOD Intraoperative swabs used within the surgical field were collected by a surgical assistant, also in charge of weighing and washing them. The liquid was collected by the aspiration system of a recuperation-washing machine (RWM). The salvaged red blood cells were collected and retransfused at the end of surgery. Several samples of the washing liquid of the swabs and salvaged blood were taken during the procedure. The correlation between the quantity of blood shed and salvaged was calculated. The biological and clinical tolerance of the transfusion was assessed. RESULTS Twelve patients undergoing surgery for scoliosis have been included. An average of 278 mL of blood were salvaged. In the washed cell concentrates the haematocrit was 54% and the free haemoglobin concentration was 3.84 g.L-1. All the bacteriological tests were negative over the first 24 hours. CONCLUSION Provided that a strict operatory protocol is followed, this study demonstrates the possibility of recuperating blood from swabs used during major orthopaedic surgery.

The effects of the infusion of a human recombinant von Willebrand factor (vWF) preparation in pigs homozygous for von Willebrand disease (vWD) were evaluated on serial measurements of von Willebrand factor antigen and activity, FVIII activity, vWF multimer analysis, in-vivo bleeding time and platelet adhesion and thrombus formation on collagen at high shear rates in an ex-vivo model of experimental thrombosis. Plasma-derived human and porcine vWF were used for comparison. Before infusion, the pigs were characterized by undetectable plasma vWF levels, a low level of FVIII, prolonged bleeding time, severely impaired platelet adhesion and thrombus formation. After infusion of the human recombinant vWF, in-vivo recovery of vWF activity ranged from 58% to 82%, depending on the dose infused, and its half-life was longer than for the plasma-derived concentrates. The highest-molecular-weight forms of human recombinant vWF were removed from the circulation gradually. Infusion of the three vWF concentrates produced inconsistent effects on bleeding time and moderate improvement of platelet adhesion and thrombus formation. After infusion, a prolonged increase of FVIII (> 48 h) was observed, suggesting that human recombinant vWF is able to bind and to stabilize porcine factor VIII and that porcine vWD is a good model for studying such interactions.