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We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the alpha(v)beta(3) integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG-VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG-VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against gamma-glutamyl-epsilon-lysine isopeptide bond. tTG -VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG-VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% (P0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.

Objective To compare the effect of aspirin and enoxaparin on live births in women with unexplained recurrent miscarriages, as well as secondary outcomes including birth weight, uterine and umbilical blood flows, and congenital malformations. Design Multicenter randomized comparative cohort study. Setting Four centers including two university hospitals, a peripheral general hospital, and a community health clinic. Patient(s) One hundred seven patients were randomized, 104 were available for analysis; 54 were randomized to enoxaparin and 50 to aspirin. Intervention(s) Treatment with enoxaparin or aspirin in subsequent pregnancy. Main Outcome Measure(s) Subsequent live births or miscarriage, and the incidence of obstetric complications. Result(s) Both groups had a similar live birth rate (relative risk=0.92, 95% confidence interval: 0.58–1.46). In primary aborters, live births occurred in 17 of 18 (94%) enoxaparin-treated pregnancies compared to 18 of 22 (81%) aspirin-treated pregnancies. In the aspirin group, two pregnancies were terminated: for tricuspid insufficiency and for hemolysis, elevated liver enzymes, low platelet (HELLP) syndrome. One enoxaparin-treated infant was growth restricted (2,020 g) at 36 weeks. Preeclampsia was found in three aspirin-treated patients. Preterm delivery, placental Doppler blood flow, apgar scores, and mean birth weights were similar in both groups. In the aspirin group, one infant underwent orchidectomy after testicular torsion in utero, and one infant had hypoglycemia and convulsions. Conclusion(s) Both regimens were associated with a high live birth rate and few late pregnancy complications.

Factor XIII is a plasma transglutaminase that participates in the final stage of the coagulation cascade. Thrombin-activated FXIII (FXIIIa) catalyzes the formation of covalent cross-links between gamma-glutamyl and epsilon-lysyl residues on adjacent fibrin chains in polymerized fibrin to yield the mature clot. In addition to its role in hemostasis, FXIII is known to participate in wound healing and embryo implantation, which are processes involving angiogenesis. In this review, we discuss the role of FXIII in angiogenesis and the molecular mechanisms underlying its proangiogenic effects. The FXIII role in tissue repair and remodeling may at least in part be attributed to its pro-angiogenic activity.

SummaryFactor XIII that stabilizes fibrin clots in the final stages of blood coagulation also participates in wound healing,as can be inferred from a delay in wound repair in some patients with inherited FXIII deficiency. In this study we evaluated the effect of FXIII on wound healing in FXIII-deficient mice. Three groups of mice (n = 10) were employed: control group, FXIII-deficient group and FXIII-deficient group treated with FXIII concentrate. Excisional wounds were left unsutured and undressed, and mice were followed for eleven days. FXIII-deficient mice exhibited impaired wound healing as has been demonstrated by 15%, 27% and 27% decrease in percentage of wound closure on day 4, 8 and 11, respectively. On day 11 complete healing was observed in control (100% closure), 73.23% in FXIII-deficient and 90.06% in FXIII deficient/FXIII-treated groups (p = 0.007 by ANOVA and p = 0.001 by t-test between control and FXIII-deficient groups). Scoring system representing maturation rate of the wounds showed that the scores for the control,FXIII-deficient and FXIIIdeficient/ FXIII treated groups were 94.9 ± 4.7, 61.5 ± 14.5 and 94.5 ± 6.4, respectively (p < 0.001 by ANOVA). Histological analysis of the lesions performed at day 11 disclosed delayed reepithelization and necrotized fissure in FXIII-deficient mice and normal healing in FXIII-deficient/FXIII-treated mice. The findings of this study confirm that in FXIII-deficient mice wound healing is delayed and the cellular and tissue defects can be corrected by treatment with FXIII, providing evidence for the essential role of FXIII in wound repair and remodeling.

Objective— Coagulation Factor XIII (FXIII) was previously shown by us to induce angiogenesis. The aim of this study was to elucidate the molecular events underlying the proangiogenic effects of activated FXIII (FXIIIa) on human umbilical vein endothelial cells (HUVECs). Methods and Results— As shown by coimmunoprecipitation studies, FXIIIa crosslinked α v β 3 with vascular endothelial growth factor receptor 2 (VEGFR-2) and enhanced the noncovalent interaction between the 2 receptors. In addition, FXIIIa induced tyrosine phosphorylation of VEGFR-2 in both the crosslinked high-molecular-weight and the noncovalent VEGFR-2/α v β 3 complexes. These effects as well as FXIIIa-induced proliferation and migration of HUVECs were abolished by iodoacetamide treatment of FXIIIa (I-FXIII) or by PTKI, an inhibitor of VEGFR-2. FXIIIa induced upregulation of c-Jun and Egr-1 as revealed by quantitative RT-PCR. Electrophoretic mobility-shift assay experiments showed that FXIIIa treatment of HUVECs enhanced binding of Wilm’s tumor-1 (WT-1) but not of early growth response (Egr)-1 to the thrombospondin-1 (TSP-1) promoter sequence, suggesting that WT-1 but not Egr-1 is involved in downregulation of TSP-1 expression. Conclusions— The proangiogenic effect of FXIIIa is mediated by (1) enhancement of crosslinked and noncovalent α v β 3 /VEGFR-2 complex formation; (2) tyrosine phosphorylation and activation of VEGFR-2; (3) upregulation of c-Jun and Egr-1; and (4) downregulation of TSP-1 induced indirectly by c-Jun through WT-1. These processes may clarify FXIII role in vascular remodeling and tissue repair.

The effect of thromboprophylaxis with low molecular weight heparin (LMWH), on the subsequent live birth rate, in thrombophilic women with recurrent miscarriage has not been sufficiently assessed. The present study is a cohort study undertaken to assess the effect of enoxaparin on the subsequent live birth rate in women with hereditary thrombophila. Eighty-five patients with three or more consecutive pregnancy losses and a hereditary thrombophilia subsequently conceived. Thirty-seven were treated with daily subcutaneous injections of enoxaparin 40 mg and 48 were not treated. The outcome of the subsequent pregnancy was assessed in both groups of patients in terms of live births or repeat miscarriage. Forty-seven of the 85 patients were subsequently delivered, 38 have miscarried. Twenty-six of the 37 pregnancies in treated patients (70.2%) resulted in live births, compared with 21 of 48 (43.8%) in untreated patients (P < 0.02, OR 3.03, 95% CI 1.12-8.36). The beneficial effect was seen mainly in primary aborters, i.e. women with no previous live births (P < 0.008, OR 9.75, 95% CI 1.59-52.48). This benefit was also found in patients with a poor prognosis for a live birth (five or more miscarriages), where the live birth rate was increased from 18.2% to 61.6%. However, the benefit was not statistically significant, probably due to the small number of patients. If the beneficial effects of enoxaparin are confirmed by additional studies, thromboprophylaxis can be recommended for patients with hereditary thrombophilia and recurrent pregnancy loss.

Of all the coagulation factor deficiencies, only deficiency of factor XIII (FXIII) and fibrinogen are associated with pregnancy loss. FXIII deficiency and a complete or partial deficiency of fibrinogen are associated with bleeding since childhood, impaired wound repair, and recurrent spontaneous abortions. Both FXIII and fibrinogen play an essential role in placental implantation and maintenance of pregnancy. The causes of miscarriages in FXIII or fibrinogen-deficient women is discussed and an appropriate treatment advocated.

We have designed a simple test for the early diagnosis and treatment monitoring of thrombotic thrombocytopenic purpura (TTP). We examined plasma from 24 TTP patients and normal plasma using a cone and plate(let) analyser (CPA). Test plasma was mixed with citrated normal whole blood (group O) and subjected to flow at a shear rate of 1800/s. Mixing normal plasma (12.5, 25, 50 or 75 microl) with heterologous normal whole blood (final volume of 200 microl) resulted in a decrease of surface coverage (SC, maximally by 63%) and, to a lesser extent, of average size (AS, maximally by 37%) due to dilution of the blood sample. In contrast, mixing the same quantities of acute TTP plasma with normal blood yielded an increase in both SC (up to 125%) and AS (up to 130%). Increased SC and/or AS were detected in all 15 patients in acute phase and in three out of 14 patients in remission. Following repeated plasmapheresis, the enhanced platelet deposition in five patients with acute TTP returned to almost normal patterns. Mixing plasma from patients with other thrombocytopenic conditions in this way resulted in a decrease in both SC and AS, and did not differ from control subjects. In conclusion, the CPA is a simple and specific laboratory test that can be used for the diagnosis and monitoring of plasma exchange therapy in TTP.

Objective: To assesses the live birth rate without treatment in women with hereditary thrombophilia who have recurrent miscarriage and women without thrombophilia who have recurrent miscarriage. Design: Prospective observational study. Setting: Tertiary referral unit in university hospital. Patient(s): One hundred twenty women with thrombophilia and 65 women without thrombophilia. Main Outcome Measure(s): Number of live births or repeated miscarriages. Results: Of the 185 patients, 44 with thrombophilia and 26 without thrombophilia have conceived. Nineteen of the 44 pregnancies (43.2%) in thrombophilia patients have terminated in live births, compared with 8 of 26 pregnancies (30.8%) in patients without thrombophilia. This difference is not statistically significant. Conclusions: Hereditary thrombophilia did not seem to affect the live birth rate in women with recurrent miscarriage.

Platelet adhesion and aggregation are mediated by specific platelet membrane glycoproteins GPla/IIa, GPlb alpha, and GPIIb/IIIa, and are essential steps in thrombus formation and development of acute myocardial infarction.To evaluate the risks exerted by each of the following polymorphisms in young males with AMI: HPA-1a/b in GPIIIa: 807C/T in GPIa; and HPA-2a/b. VNTR and Kozak C/T in GPlb alpha.We conducted a case-control study of 100 young males with first AMI before the age of 53 and 119 healthy controls of similar age. All subjects were tested for the above polymorphisms.The allele frequencies of each of the platelet polymorphisms were not significantly different between the young man with AMI and the controls. Smoking alone was associated with a 9.97-fold risk, and the presence of at least one metabolic risk factor resulted in a 2.57-fold risk of AMI.The platelet glycoproteins polymorphisms studied are not an independent risk factor for AMI.

Coagulation factor XIII (FXIII) is a transglutaminase that catalyzes crosslink formation in fibrin clots. Endothelial cells (EC) were demonstrated to bind FXIII via their alpha(v)beta3 integrin receptor. FXIII was also shown to bind platelet glycoprotein IIb/IIIa receptor. In the present study, we analyzed if FXIII can mediate platelet-EC interaction. Both FXIII and activated FXIII (FXIIIa) bound to EC monolayers; this binding was enhanced by the addition of Mn2+ and was inhibited by the monoclonal antibody L609 against alpha(v)beta3 integrin. Normal washed platelets also bound surface-immobilized or soluble FXIII and FXIIIa, and the binding was GPIIb/IIIa dependent. The effect of FXIII concentrate (Fibrogammin-P) treatment on the interaction of ECs with platelets from six FXIII-deficient patients was studied. Patients' platelets were radiolabeled with 3H-Adenine, washed, resuspended in autologous plasma and allowed to adhere to immortalized EC line EAhy926. Adhesion of platelets from FXIII-deficient patients to ECs increased 1.7+/-0.4-fold (P=.01) following intravenous infusion of FXIII concentrate. Similarly, addition of 1 U/ml of FXIII concentrate to the patients' PRP in vitro increased the adhesion 1.8+/-0.5-fold (P=.008). Preincubation of the EC monolayers with increasing concentrations of either FXIII or FXIIIa augmented the adhesion of normal washed platelets to ECs in a dose-dependent manner. At 10 U/ml of EC-bound FXIII or FXIIIa, platelet adhesion enhanced 1.7+/-0.25-fold (P=.03) and 2.5+/-0.5-fold (P=.02), respectively. The increase in platelet adhesion was completely abolished by pretreatment of ECs with the anti-alpha(v)beta3 antibody L609 or by preincubation of the platelets with the GPIIb/IIIa inhibitor Abciximab. Taken together, our data indicate that FXIII mediates the interaction of platelets with ECs by bridging between endothelial alpha(v)beta3 and platelet GPIIb/IIIa integrins. This interaction may be relevant for tissue remodeling and wound repair after vascular injury in FXIII-deficient patients.

Objective: To determine the prevalence of markers of thrombophilia in women hospitalized for severe OHSS. Design: Prospective study. Setting: Academic research center. Patient(s): Women undergoing induction of ovulation complicated by severe OHSS (n = 20) and women undergoing induction of ovulation without development of severe OHSS (n = 41). Intervention(s): Blood samples to test for markers of thrombophilia were obtained during the luteal phase of the treatment cycle. Main Outcome Measure(s): Blood samples were analyzed for markers of thrombophilia, such as plasma levels of antithrombin, protein S and protein C, antiphospholipid antibodies, the factor V Leiden mutation, and 677T polymorphism in the 5,10 methyltetrahydrofolate reductase ( MTHFR 677T) gene. Result(s): Seventeen of 20 patients with severe OHSS (85%) and 11 of 41 controls (26.8%) had one or more positive markers of thrombophilia. Of the women with severe OHSS, 6 had a decreased antithrombin level, 8 had decreased levels of protein S, 7 were homozygous for the MTHFR 677T mutation, 1 was heterozygous for the factor V Leiden mutation, and 5 had antiphospholipid antibodies. Eight women with OHSS and no controls had more than one positive marker of thrombophilia. Conclusion(s): The prevalence of thrombophilia is increased in women with severe OHSS. These findings suggest that prophylactic screening for this disorder and possible use of heparin prophylaxis for thromboembolic phenomena should be considered in these patients.

Background and Purpose — Although risk factors for carotid artery stenosis caused by atherosclerosis are known, it is unclear what triggers “activation” of the atherosclerotic plaques and the ensuing thromboembolic cerebral events. The aim of this study was to evaluate whether thrombophilic factors, platelet glycoprotein (GP) polymorphisms, and homocysteine are associated with a risk of ischemic events in patients with significant carotid stenosis. Methods — Consecutive patients with ≥50% carotid stenosis, whether symptomatic (with ipsilateral ischemic events) or asymptomatic, who were evaluated and followed in a neurovascular clinic were tested for plasma levels of homocysteine, C677T mutation in methylenetetrahydrofolate reductase, G20210A mutation of factor II, factor V Leiden, antiphospholipid antibodies, and polymorphisms of platelet membrane GP: human platelet antigen (HPA)-1, GP Ia (C807T), and GP Ib (variable number of tandem repeats, Kozak, and HPA-2). Results — Eighty-six asymptomatic and 67 symptomatic patients were evaluated. The former group was older (73.7±6.9 versus 69.5±9.1 years, P =0.02). Major risk factors for stroke were similar in both groups. In symptomatic patients versus asymptomatic patients, hyperhomocysteinemia was 3-fold more frequent (34.3% versus 12.8%, respectively; P =0.002) and HPA-1a/b was almost 2-fold more common (38.8% versus 20.9%, respectively; P =0.01). All other thrombophilic factors and platelet polymorphisms studied did not differ significantly between the 2 groups. Multivariate analysis revealed that hyperhomocysteinemia and the HPA-1a/b genotype conferred a significant risk of cerebral ischemic events, with odds ratios (95% CI) of 4.07 (1.7 to 9.7) and 3.4 (1.5 to 7.8), respectively. Conclusions — Hyperhomocysteinemia and HPA-1a/b are independent risk factors for ischemic events in patients with significant carotid stenosis.

BAT is a monoclonal antibody produced against membranes of Daudi cells that induces anti-tumor activity in mice against a variety of solid murine and human tumors, mediated by its immune stimulatory properties on murine and human lymphocytes. The present study analyzes the effect of BAT on leukemia/lymphoma using the BCL1 model of leukemia/lymphoma in BALB/C mice. BAT antibody binds to BCL1 leukemia cells and recognizes a 48 kDa protein similar to the antigen on Daudi cells. Mice inoculated with leukemia cells were treated either by direct BAT injections or by adoptive transfer of lymphocytes from BAT-injected mice. Administration of BAT monoclonal antibody was either once, on day 14, or daily on days 10-13 post tumor inoculation. A single injection of BAT resulted in reduction of peripheral blood tumor cells, however additional injections further decreased the tumor cell number reaching a 95-fold reduction on day 20 post tumor inoculation. Anti-tumor effect was also obtained when animals were injected with splenocytes from BAT-treated donor mice. A significant prolongation of survival of BAT-treated mice was observed although with no cure. The results of this study indicate that BAT might be used for reducing the tumor burden in leukemia for immunotherapy and in combination with other treatment modalities.

SummaryAcquired von Willebrand syndrome (AVWS) has been associated mainly with monoclonal gammopathy of uncertain significance (MGUS), clonal lymphoproliferative or myeloproliferative disorders and autoimmunity. In the present work we studied 6 patients with AVWS: four with MGUS IgG (λ or κ), one with small lymphocytic lymphoma and one with agnogenic myeloid metaplasia (AMM). All the patients underwent a pharmacokinetic analysis at presentation in order to study potential differences in recovery, clearance (CL) or terminal half-life (THL) following administration of von Willebrand factor (VWF) concentrate. In all the patients with AVWS an increase in clearance and a decrease in THL was observed as compared to these parameters in patients with hereditary type 3 von Willebrand disease (VWD). No difference in recovery was observed among the groups. The increase in clearance and the decrease in THL were significantly more pronounced in the group of MGUS patients (57.93 ± 25.6 ml/h/kg, and 1.39 ± 0.5 h, respectively) as compared to these parameters in the AMM (8.06 ml/h/kg, and 6.96 h, respectively) or the lymphoma (4.76 ml/h/kg, and 6.76 h, respectively) patients (p = 0.03 for clearance and 0.001 for THL). These data indicate that the pharmacokinetic analysis can be a useful tool to distinguish between MGUS-related and other causes of AVWS, and to plan an appropriate treatment accordingly.

Background and Purpose —The association between ischemic childhood stroke and thrombophilia has been debated. We studied the prevalence of thrombophilia risk factors in 65 unrelated children with ischemic stroke compared with 145 control subjects. Methods —Patients and control subjects were tested for antithrombin protein C and protein S deficiencies, the presence of antiphospholipid antibodies (APLA), factor V Leiden (FVL), G20210A polymorphism of factor II gene (FII G20210A), and C677T polymorphism of 5,10-methylenetetrahydrofolate reductase gene (C677T MTHFR). Results —Of 65 children, 7 had a stroke in the neonatal/perinatal period and therefore were analyzed separately. Thirty-one of the remaining 58 patients with pediatric stroke (53.4%) were found to have at least 1 thrombophilia marker compared with only 25.5% of control subjects. None of the patients or control subjects had protein S or antithrombin III deficiency. The prevalence of protein C deficiency was higher among pediatric stroke patients than among control subjects, but the difference was not statistically significant (OR=7, 95% CI 0.75 to 65.1). Heterozygous FII G20210A and homozygous MTHFR 677T were not associated with an increased risk for stroke (OR=1.29, 95% CI 0.2 to 8.2; and OR=1.06, 95% CI 0.4 to 2.7, respectively). In contrast, the presence of APLA was associated with a >6-fold risk of stroke (OR=6.08, 95% CI 1.5 to 24.3), and the heterozygosity for FVL increased the risk of stroke by almost 5-fold (OR=4.82, 95% CI 1.4 to 16.5). Five patients with pediatric stroke had a combination of ≥2 thrombophilia markers, whereas none of the control subjects had a combination of the markers. Most of the patients with neonatal/perinatal stroke were found to have at least 1 thrombophilia marker. Conclusions —These data suggest that the prevalence of thrombophilia markers is increased in children with stroke compared with control subjects and, specifically, that FVL and APLA contribute significantly to stroke occurrence.

Hyperhomocysteinemia is a defined risk factor for venous thromboembolism (VTE). Several polymorphisms of genes encoding for enzymes acting in the remethylation pathway of homocysteine metabolism, ie, methionine synthase (MS) A2756G, methylenetetrahydrofolate reductase (MTHFR) C677T and MTHFR A1298C, can cause increased homocysteine levels particularly in patients with deficiencies of folic acid, vitamin B6, or B12 and hence be potential risk factors for VTE. Indeed, homozygous MTHFR C677T was shown to be a mild risk factor for VTE by some, but not by all, investigators. In this study, we assessed the risk exerted by MS A2756G and MTHFR A1298C in a cohort of patients with idiopathic venous thromboembolism. Homozygosities for MS A2756G and MTHFR A1298C were not found to be statistically significant risk factors for VTE. In addition, no interactions were observed among MS A2756G, MTHFR A1298C and MTHFR C677T in conferring a risk of VTE.

Abstract —Platelet adhesion to exposed subendothelium is mediated by platelet receptor glycoprotein Ib and polymeric von Willebrand factor (vWF). To improve the results of coronary arterial thrombolysis, fragments of vWF with enhanced glycoprotein Ib binding competitive with native vWF have been proposed as adjuvants to recombinant tissue-type plasminogen activator (rtPA). We designed a recombinant vWF fragment spanning Ala444 to Asp730 that contains the Arg545Cys mutation (named AR545C) and analyzed its antiplatelet properties in vitro and in vivo. AR545C-platelet interaction was assessed by ristocetin or botrocetin-induced platelet agglutination, or interaction with extracellular matrix under arterial flow conditions. AR545C showed enhanced reactivity with platelet glycoprotein Ib at low concentrations of ristocetin, and 60% bound spontaneously to platelets. AR545C inhibited ristocetin-induced platelet agglutination in a dose-dependent manner, with a concentration necessary to inhibit 50% of agglutination of 0.16±0.04 μmol/L. The inhibitory effect of AR545C on rabbit botrocetin-induced platelet agglutination was also dose dependent, with a concentration necessary to inhibit 50% of agglutination of 0.3 to 0.5 μmol/L. AR545C also completely inhibited aggregate formation and decreased the adhesion of platelets to extracellular matrix by 62.5%. The effect of AR545C on thrombolysis with rtPA was evaluated using a modified rabbit femoral thrombosis model. Local injection of AR545C into the thrombosed segment of rabbit femoral artery significantly shortened the time to reperfusion with rtPA (60±17.3 versus 103±15.2 minutes, P =.05) and significantly prolonged the total patency time (175 versus 21 minutes, P =.04). No significant difference was found in the reperfusion rate or time to reocclusion. AR545C is a potential antithrombotic agent that enhances the thrombolytic effect of rtPA in the rabbit model.

We describe 2 sibs (brother and sister) with myopathy, sideroblastic anemia, lactic acidosis, mental retardation, microcephaly, high palate, high philtrum, distichiasis, and micrognathia. Very low levels of cytochromes a, b, and c were detected in the patients' muscle mitochondria. Deposition of iron within the mitochondria of bone marrow erythroblasts was observed on electron microscopy. Irregular and enlarged mitochondria with paracrystalline inclusions were also seen on electron microscopy of the patients' muscle specimen. Examination of DNA from the affected sibs showed no deletions in the mitochondrial DNA nor the mutations identified in the syndromes of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) or myoclonus, and epilepsy associated with rugged-red fibers (MERRF). Since the parents were first cousins and 2 of 6 sibs (male and female) were affected, we suggest that the syndrome expressed by our patients represents a previously unknown autosomal recessive disorder that includes mitochondrial myopathy, lactic acidosis, and sideroblastic anemia. © 1995 Wiley-Liss, Inc.

Abstract 2665 Background: Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells. Aim: To study the role of FGL-2 in patients with lymphoproliferative disorders. Our hypothesis is that upregulation of FGL-2 activity in patients with B-cell malignancies may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of malignant cells. Methods: Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from 29 patients with active lymphoproliferative disorders and 107 normal controls. Informed consent was obtained from every participant. PBMC extracts were incubated with an equal volume of human prothrombin (final concentration10 μM) for 30 min at 37 °C. Thrombin generation was measured at 405 nm using an automated plate reader after addition of chromogen S-2238. The thrombin activity of each sample was calculated by comparison with absorbance curve generated by known concentrations of human thrombin. FGL-2 was immunoprecipitated (IP) from PBMC with an anti-FGL-2 antibody and EZview Red Protein A Affinity Gel (sigma # P6486). The activity of IP FGL-2 was measured by thrombin generation assay. The expression of FGL-2 was analyzed in HUVEC and PBMC in the presence or absence of IF-γ at 20 ng/ml. Total RNA was isolated using RNAqueous™ (Ambion #AM1912) and RT-PCR, analysis was performed using Rotor-gene RG-3000 (Corbett). The difference in cycle time (ΔCT) was measured by comparing FGL-2 gene with ABL-1 gene (house keeping gene). The relative quantification was calculated by the formula RQ= 2−ΔCT. HUVEC were transfected with 0.5 μM SiRNA synthesized complementary to FGL-2 (Target SiRNA) using Dharmacon ON-TARGET plus SMART pool reagent (Thermo Fisher Scientific) according to manufacturer instructions. FGL-2 expression following addition of target SiRNA was compared to that obtained following addition of non-specific (non-target) SiRNA. Student's t-test was used for all comparisons. Results: As shown in the table, almost 3-fold increase in FGL-2 activity in PBMC was observed among patients with active B-cell lymphoma, either aggressive or indolent, as compared to that of healthy controls (p Three-fold increase in thrombin generation was obtained in PBMC from the patients following IP, similarly to that observed in non-IP PBMC. A significant increase in mRNA of FGL-2 was found in either PBMC of lymphoma patient or HUVEC treated with IF-γ. In HUVEC the increase in FGL-2 mRNA was inhibited by 80% following treatment with specific SiRNA. Conclusions: Disclosures: No relevant conflicts of interest to declare.

Abstract 20 Congenital Factor XIII (FXIII) deficiency is a rare autosomal recessive disorder affecting approximately 1 in 2–5 million individuals without gender or ethnic predilection. The plasma form of FXIII is a heterotetramer [A2B2] comprising two FXIII-A subunits and two FXIII-B subunits. A-subunit deficiency predominates (95%). FXIII deficient patients have a high risk of life-threatening bleeds, notably spontaneous intracranial haemorrhage. They experience impaired wound healing, and recurrent first trimester spontaneous abortions. Given the severity of the disorder there is a clinical need for providing effective haemostatic replacement therapy. Currently, only plasma derived products are approved for use in the United States. These may carry a risk of blood borne infection, sensitization and allergic reactions. A multi-center, multi-national, open-label, single-arm, multiple dosing phase 3 (prophylaxis) trial was undertaken to evaluate the efficacy and safety of a novel recombinant FXIII (rFXIII) in the prevention of bleeds in congenital FXIII-A subunit deficiency. This rFXIII-A2 dimer is expressed in Saccharomyces Cerevisiae and is an exact copy of the human A-subunit, which is the active cross-linking enzyme. When rFXIII is administered, it immediately binds to the B subunit that is present in the bloodstream. Forty-one patients aged ≥7 years [mean: 26.4 (range: 7–60); 23 males; 18 females] with a diagnosis of severe congenital FXIII-A subunit deficiency were enrolled. Patients entered a 4-week run-in period followed by monthly treatment with 35 IU/kg of rFXIII for 52 weeks. Informed consent was obtained prior to any trial-related activities. Bleeding episodes and adverse events were recorded. Testing was conducted monthly to measure pre- and post-dose FXIII activity and concentration, 5M urea clot solubility and anti-FXIII antibodies. Where antibodies were found, detailed testing for neutralizing potential was performed. Data were compared with bleeding rates based on historical data. During the treatment period with rFXIII (466 patient months) five bleeding episodes treated with FXIII-containing products were observed in four patients. All five were associated with trauma. No spontaneous bleeds that require treatment or intracranial haemorrhage occurred during rFXIII treatment period in any of the patients. The annual rate of bleeds requiring treatment was estimated via a Poisson model to be 0.048 bleeds/patient/year (95% CI: [0.0094; 0.2501] - significantly lower than the historic bleeding rate of 2.91 (p Disclosures: Inbal: NovoNordisk: Honoraria, Research Funding. Oldenburg:novonordisk: Research Funding. Carcao:NovoNordisk: Research Funding. Rosholm:NovoNordisk: Employment. Tehranchi:NovoNordisk: Employment. Nugent:NovoNordisk: Consultancy, Research Funding.

Endothelial cells (EC) were cultured from the umbilical cord of a male neonate whose mother was previously diagnosed with type IIA von Willebrand's disease (vWd). The diagnosis of type IIA vWd in the proband was confirmed by low ristocetin activity and the absence of the highest molecular weight (MW) forms of von Willebrand factor (vWf) in his platelet poor plasma. The vWf of EC cultured from the neonate's umbilical cord differed from that of control EC and the cell line EA.hy926 in two respects. Firstly, the full range of molecular weight forms was present in the patient EC lysate and, secondly, vWf:Ag expression was approximately seven-fold greater than that of control cells. Platelet lysates prepared from other affected members of the type IIA vWd family in the presence or absence of proteolytic inhibitors demonstrated a near normal vWf multimeric distribution. Resistance of these high MW forms to heat degradation was conferred by the presence of proteolytic inhibitors. Moreover, the full plasma vWf multimeric distribution could not be restored by the inclusion of EDTA. N-ethylmaleimide and leupeptin in the anticoagulant during the rapid preparation of platelet poor plasma. These findings lend support to the heterogeneous nature of type IIA vWd and has possible implications in the understanding of the intracellular processes involved in the biosynthesis and storage of the vWf macromolecular complex as well as the pathogenesis of type IIA vWd.

Glanzmann’s thrombasthenia is an autosomal recessive bleeding disorder characterized by severely reduced or absent platelet aggregation in response to physiologic agonists because of quantitative or qualitative abnormalities of platelet glycoprotein IIb/IIIa (GpIIb/IIIa). Acquired Glanzmann-like thrombasthenia may develop as a result of autoantibodies specific to platelets GpIIb/IIIa and has been described in association with autoimmune disorders. Patients with the acquired form experience variable degrees of mucocutaneous bleeding, and their platelets fail to aggregate with agonists such as ADP, thrombin, collagen, or epinephrine, whereas ristocetin-induced aggregation is normal. Treatment consists of a combination of one or more of the following: corticosteroids, chemotherapy, plasma exchange and high dose intravenous immunoglobulin (IVIG). We present a 75-year old woman with SLE who developed ITP at the age of 60 and underwent splenectomy with subsequent normalization of platelet counts. However, since that operation, the patient has been admitted several times due to variable degrees of mucocutaneous bleeding, including one episode of massive gastrointestinal hemorrhage. Platelet counts were 184 to 284 X 103/μL. Laboratory evaluation revealed a bleeding time of 15 minutes (normal 0.5 to 8 minutes) and no aggregation of platelets in response to ADP, epinephrin, collagen, and thrombinbut normal ristocetin-induced aggregation. Addition of 25% of patient’s platelet poor plasma to normal whole blood revealed reduced platelet deposition by 61% using cone and plate analyzer, thus, confirming the diagnosis of acquired Glanzmann’s thrombasthenia due to autoantibodies. Because of treatment failure with corticosteroids, immunosuppession and IVIG, anti-CD20 monoclonal antibody (Rituximab) 375 mg/kg/week for 4 weeks was administered resulting in normalization of platelets aggregation and complete clinical remission. Anti-CD20 therapy might be a therapeutic option for intractable antibody-mediated acquired thrombasthenia.

FXIII, the last factor of the coagulation cascade, is a plasma transglutaminase that stabilizes fibrin clots by cross-linking fibrin chains. In addition to its role in coagulation, FXIII is essential for embryo implantation and wound healing - processes that involve angiogenesis. In our previous work (Dardik et al., Arterioscler. Thromb. Vasc. Biol., 2003:23: 1472) we showed that thrombin-activated FXIII (FXIIIa) exhibits pro-angiogenic activity that is associated with downregulation of thrombospondin (TSP-1). We next shoowed that the proangiogenic effect of FXIIIa is mediated by the phosphorylation and activation of VEGFR-2 receptor. Activated VEGFR-2 then initiates a signaling cascade leading to upregulation of cyclin D-1, Egr-1 and c-Jun, and downregulation of TSP-1 induced by c-Jun via WT-1 (submitted). In the current study we evaluated the pro-angiogenic effect of FXIIIa on cardiac ischemia (using rat myocardial ischemia model) or transplantation (usig murine neonatal cardiac allograft into pinnae ear transplant model). In addition, angiogenesis in FXIII-deficient mice was studied using in vivo Matrigel model. Following myocardial ischemia, a significant increase in new vessel formation was observed in the FXIIIa-injected as compared to saline-injected rats: 142±15/mm2 vs 64±15/mm2; respectively, p = 80% beating area was significantly higher in the FXIIIa-treated group that in the control: 46% vs 17%, respectively; p = 0.038. Histological analysis of the matrigel sections from FXIII-deficient mice showed significantly decreased number of new vessels compared to that of the control mice and the number of the vessels increased after treatment with FXIII (5.9±1.9 vr. 8.8±2.4 vr. 7.4±2.9, respectively, P=0.004). Similarly, the values of hemoglobin measured in the matrigel tissue were significantly higher in the control group as compared to that of the FXIII-deficient - group, increasding after treatment with FXIII (4.6 ± 2.5 mg/mg matrigel vs 1.3 ± 1.0 mg/mg matrigel vs. 4.4±0.6 mg/mg matrigel, respectively; p = 0.01. The results of this study confirm the pro-angiogenic activity of FXIIIa in ischemic tissues and clarify its role in tissue remodeling, thus, opening new therapeutic options for this compound with regard to clinical conditions where perfusion via development of new vessels is crucial.

von Willebrand protein was found to promote the incorporation of platelets into evolving fibrin thrombi. Using formalin-treated or fresh platelets, both the initial rate and extent of platelet incorporation into polymerizing fibrin were dependent on von Willebrand protein. von Willebrand protein was incorporated into evolving fibrin thrombi in parallel with platelets. Soluble fibrin monomer covalently linked to acrylonitrile beads (Matrex 102) bound von Willebrand protein specifically and saturably with an apparent approximate dissociation constant (KD) of 15 micrograms/ml. Glycocalicin, the water-soluble proteolytic fragment of glycoprotein Ib, bound to fibrin monomer in this system specifically and saturably, as well, with an apparent approximate KD of 5 micrograms/ml, but only in the presence of saturating concentrations of von Willebrand protein. These data demonstrate that the initial rate and extent of platelet incorporation into evolving fibrin thrombi are dependent on von Willebrand protein; von Willebrand protein serves as a link between polymerizing fibrin and platelet surface glycoprotein Ib; and von Willebrand protein binds to fibrin monomer and is thereby able to bind to platelet surface glycoprotein Ib in the absence of ristocetin.

Pseudoxanthoma elasticum (PXE) is a systemic disease that affects the elastic fibers of the arterial walls producing dilatation, occlusion and eventually rupture of arteries. The lowering of visual acuity in patients harboring PXE is currently thought to account for the presence of fibrovascular ingrowth into the angioid streaks. The authors saw a 46-year-old patient with bilateral lowering of visual acuity due to bilateral anterior ischemic optic neuropathy (AION) but in whom uncomplicated angioid streaks, PXE and an increased platelet agglutination were found.

A patient with an unusually prolonged course of Ph' positive chronic myeloid leukemia is presented. His disease was marked by cyclic leukocytosis, various chromosomal aberrations and secondary thrombasthenia. In vitro culture studies and granulocyte-macrophage colony stimulating factor (GM-CSF) production were consistent with responsiveness of the leukemic clone to GM-CSF. The possible relationship between the long survival and the feedback regulation of leukopoiesis is raised.

The activity of lymphocyte uroporphyrinogen synthase (URO-S) was examined in 51 non-Hodgkin's lymphoma (NHL) patients at various follow-up periods. Mean +/- SD activity (pmol porphyrin/mg protein/hr) at diagnosis (n = 24), on relapse (n = 14) and during active disease (n = 14) were 31.7 +/- 19.8, 31.7 +/- 27.2 and 29.4 +/- 18.5, respectively. These values were significantly higher than the enzyme activity during remission (14.1 +/- 4.0), which was in the normal range (14.5 +/- 3.8). Abnormally high activity was found in 65.4% of determinations at diagnosis, on relapse and during active disease, compared to 5.5% during remission (P less than 0.001). Significant association of abnormal URO-S activity was found with advanced clinical stage (P less than 0.01), spleen enlargement (P = 0.048), involvement of bone marrow (P = 0.02), as well as lymphoma cell spread to peripheral blood (P = 0.03). Highly significant correlation (r = 0.65, P less than 0.001) was found between URO-S activity and serum lactic dehydrogenase (LDH) levels. Excessively high levels of URO-S activity were found only in patients with lymphoma cells in peripheral blood. No association was found with histopathologic classification and liver size. The authors conclude that URO-S activity is a biochemical indicator for patients in all stages of NHL and seems to be a specific marker for the extensiveness of the disease.

We describe a patient with hemophagocytic syndrome resembling malignant histiocytosis which was complicating myelodysplastic disease of 3 years duration. Detailed morphological and ultrastructural studies indicate that the histiocytic component did not demonstrate features of malignancy. A review of other known malignancies ending up in the hemophagocytic syndrome is given, and the significance of this syndrome is discussed.

The recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC50 = 0.02 ± 0.006 μmol/L v 0.1 ± 0.03 μmol/L, P = .001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate–induced platelet aggregation (IC50 = 0.018 ± 0.002 μmol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 μmol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P ≤ .004 for each parameter by ANOVA). In an ex vivo rabbit model, we also found that S-nitroso-AR545C had a more marked and more durable inhibitory effect on botrocetin-induced platelet aggregation than did AR545C, and these differences were also reflected in the extent and duration of effect on the prolongation of the bleeding time in these animals. These data show that S-nitroso-AR545C has significant and unique antiplatelet effects, inhibiting both adhesion and aggregation, by blocking platelet GPIb receptor through the AR545C moiety and elevating platelet cyclic 3′,5′-guanosine monophosphate through the -SNO moiety. These observations suggest that this NO-modified fragment of vWF may have potential therapeutic benefits as a unique antithrombotic agent.

SummaryAntiplatelet drugs are the mainstays of therapy for acute and chronic cardiovascular diseases. S-nitroso-AR545C – an S-nitrosoderivative of a recombinant von Willebrand factor fragment AR545C spanning Ala 444 to Asp 730 and containing an Arg 545 Cys mutation, was previously found to inhibit ristocetin-and ADP-induced platelet aggregation and the interaction of platelets with extracellular matrix (ECM). In the current study we tested the antithrombotic properties of S-nitroso-AR545C on guinea pig platelets and in a platelet-rich thrombosis model in the guinea pig. Preincubation of guinea pig platelets with 0.1 µM of S-nitroso-AR545C decreased ristocetin-induced agglutination by 40% (p = 0.009) and completely abolished ADP-induced aggregation (p