Dimitrios A. Tsakiris

β3-integrin–deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease

BACKGROUND Vascular endothelial cells form the interface between recipient tissues and circulating alloreactive donor T cells after allogeneic stem cell transplantation. Vascular injury has been seen in patients with acute graft versus host disease (GVHD) in the skin. We aimed to see whether vascular injury mediated by cytotoxic T lymphocytes and microvessel loss arises in patients with chronic GVHD. METHODS We investigated eight patients with acute GVHD and ten with chronic GVHD for signs of endothelial injury and microvessel loss by measurement of von Willebrand factor (vWF) in plasma and blood vessel density in biopsy samples taken from lesional skin. Controls consisted of nine patients without GVHD who survived for longer than 100 days and nine healthy people. Inflammation and endothelial injury were assessed in selected samples by immunostaining for CD8 T cells, activated cytotoxic T lymphocytes, and vascular endothelial cells. FINDINGS We identified more extensive loss of microvessels in the skin of patients with GVHD (median 66 capillaries/mm(2); IQR 16-98) than of healthy controls (205 capillaries/mm(2); 157-226; p=0.005). Patients with GVHD had higher concentrations of vWF (238%; 168-288) than did those without GVHD (102%; 88-118; p=0.0005). Perivascular CD8 T cell infiltrates in skin correlated with vWF plasma concentrations in patients with GVHD (p=0.01), and activated cytotoxic T lymphocytes and endothelial injury were present in these same samples. INTERPRETATION Host endothelial cells are a target of alloreactive donor cytotoxic T lymphocytes. Substantial blood vessel loss may lead to impaired blood perfusion and tissue fibrosis, the hallmark lesion of chronic GVHD.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories.

INTRODUCTION Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Intramedullary nailing and pulmonary embolism: does unreamed nailing prevent embolization? An in vivo study in rabbits.

Pulmonary embolism in reamed femoral nailing has been reported and discussed over recent years. Does an unreamed nailing technique with a solid nail prevent this rare but serious complication of intramedullary fixation? In an animal model in rabbits, we studied the pathophysiologic impact on pulmonary function and the impact on hemostasis of reamed and unreamed nailing of intact femora and tibiae, and of femoral fracture in relation to intramedullary pressure. No statistical difference of PaO2, PaCO2, and PCO2et was found in the femur whether a reamed or unreamed procedure was performed. Two of six animals with unreamed femoral nailing, one of six animal with reamed femoral nailing, and one of five animals with a femoral fracture fulfilled four of four or three of four criteria for embolization (increase of the difference of PaCO2 and PCO2et, decrease of PaO2, increase of blast cells in central-venous blood and bone marrow/fat in histologic section of the lungs and bone). Tibial nailing did not alter pulmonary function in either group. Intramedullary pressure was increased in all animals with perioperative impairment of pulmonary function (375 to 676 mbar). Analysis of the hemostatic results showed a significant difference of platelet activation in reamed versus unreamed nailing of the femur 1 hour after nailing (p < 0.01) and a significant decrease of fibrinogen and antithrombin III (p < 0.001/p < 0.01) in reamed femoral nailing. We conclude that unreamed nailing of the femur with a solid rod may also cause bone marrow embolization with alteration of pulmonary function as long as an important increase of the intramedullary pressure is generated during the nailing procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease

In the present study, the levels of soluble adhesion molecules P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and of other markers of endothelial activation or injury, such as thrombomodulin, von Willebrand factor (vWF), as well as homocysteine, were prospectively investigated in 71 patients (21 women, 50 men, age 68+/-13) with predominantly femoropopliteal peripheral arterial occlusive disease (PAOD, stage II-IV, Fontaine) before and after percutaneous transluminal angioplasty (PTA). Thirty patients (42.3%) developed restenosis within 6 months, defined as a > 50% reduction of the lumen diameter at the site of PTA. At entry in the study, 46% and 58% of all patients had higher than normal levels of soluble P-selectin and VCAM-1, respectively. Thrombomodulin (P < 0.01) measured at entry, was significantly higher in patients who developed late restenosis, with trends for higher values for P-selectin, VCAM-1 and vWF. The relative risks for developing restenosis were 2.41 (CI95%: 1.23-4.75) and 1.54 (CI95%: 0.98-2.72) for thrombomodulin and P-selectin, respectively. Soluble P-selectin and the severity of PAOD (Fontaine stage III/IV) were found to be statistically indicative factors for late restenosis in a logistic regression risk factor analysis with an overall predictive value of 72%. At 6 months, those who developed restenosis had also higher soluble P-selectin (P < 0.01), VCAM-1 (P < 0.05) and a trend for higher thrombomodulin. Homocysteine was elevated in 52% of the patients at entry but neither was it associated with higher restenosis rates nor did it correlate with the levels of thrombomodulin or the other adhesion molecules. These findings indicate that patients with PAOD have to a significant proportion, elevated levels of circulating soluble adhesion molecules and markers of endothelial activation occurring in concert with an ongoing atherosclerotic process.

Transplant-associated microangiopathy (TAM) in recipients of allogeneic hematopoietic stem cell transplants

Summary:We studied occurrence, risk factors and outcome of patients with transplant-associated microangiopathy (TAM) after allogeneic stem cell transplantation (HSCT). A total of 221 consecutive patients were transplanted between 1995 and 2002. TAM is defined as evidence of hemolysis and schistocytes in the first 100 days. Outcomes analyzed included TAM and overall survival. Of 221 patients, 68 had TAM. The cumulative incidence was 31 (25–38)% at 100 days. Patients with TAM had higher LDH, higher bilirubin, higher creatinine and more often neurologic symptoms. TAM was not associated with stem cell source, cyclosporine levels and was not more frequent in recent years. In multivariate analysis, risk factors for TAM included donor type, age, gender, ABO-incompatibility and acute graft-versus-host disease (aGvHD). In patients with TAM, 1-year survival was lower than in patients without TAM (27±18% for TAM with high schistocyte counts; 53±15% for TAM with low schistocyte counts; vs 78±7% in patients without TAM; P<0.0001). TAM was independently associated with mortality adjusting for donor type, age and aGvHD occurrence and severity. TAM is frequent after HSCT and is associated with mortality even after adjustment for aGvHD grade. Risk factors of TAM are similar to aGvHD. TAM may represent endothelial damage driven by donor–host interactions.

Role of Hemostatic Risk Factors for Restenosis in Peripheral Arterial Occlusive Disease After Transluminal Angioplasty

In a prospective study, the role of various hemostatic factors known to be associated with thrombotic risk was investigated in 71 patients with peripheral arterial occlusive disease (PAOD, stages II through IV, Fontaine; aged 68 +/- 13 years). Laboratory investigations were done before; 1, 24, and 48 hours after; and 3 and 6 months after percutaneous transluminal angioplasty (PTA). Thirty of 71 (42.3%) patients developed restenosis (> 50% reduction of the lumen diameter) at the site of PTA within 6 months, verified by color-coded duplex sonography. Significantly increased levels of thrombin-antithrombin III complexes (P < .01), prothrombin fragments 1 + 2 (P < .01), and D-dimers (P < .01) were found 1 hour, as well as 24 to 48 hours, after PTA. Fibrinogen (P < .01) and von Willebrand factor (P < .01) were significantly higher 48 hours after PTA. Restenotic patients as a whole had higher plasma fibrinogen (3.46 +/- 1.12 versus 2.95 +/- 0.62 g/L, P < .01) and C-reactive protein (25.4 +/- 46.7 versus 7.9 +/- 6.9 mg/L, P < .05) at baseline, as well as higher fibrinogen (P < .05) and prothrombin fragments 1 + 2 (P < .01) during months 3 to 6 after PTA. There was a nonsignificant tendency for higher values of von Willebrand factor (206 +/- 98% versus 184 +/- 100%, P = .2) at baseline in patients with restenosis, whereas tissue plasminogen activator, plasminogen activator inhibitor, coagulation screening tests, blood cell counts, and serum lipids showed no significant difference between the two groups. The relative risk for developing restenosis within 6 months while having high fibrinogen (> 2.8 g/L) or C-reactive protein at baseline was 2.80 (95% CI: 1.30-6.02, P < .01) and 1.96 (95% CI: 1.07-3.58, P < .05), respectively. Patients with critical limb ischemia (stage III/IV, Fontaine) had significantly higher fibrinogen and von Willebrand factor at repeated points of time, as well as significantly higher C-reactive protein and lower creatinine clearance at entry. In the logistic regression risk factor analysis, baseline plasma fibrinogen, C-reactive protein concentration, and the severity of the arterial disease were significantly predictive of restenosis. Our results indicate that high procoagulant factors and persistent thrombin generation of the hemostatic system might promote restenosis, particularly in patients with extended atherosclerosis. This finding suggests that new treatment strategies should be taken under consideration for patients with PAOD and PTA.

Pre-analytical effects of pneumatic tube transport on impedance platelet aggregometry.

Point-of-care platelet monitoring is increasingly used in cardiac patients treated with antiplatelet agents. The validity of a new assay needs to be evaluated not only for reproducible data in clinical samples, but also for other pre-analytical conditions that may influence measurements. The aim of this study was to evaluate the influence of a pneumatic tube system (PTS) for specimen transport on impedance platelet aggregometry. We evaluated 50 consecutive patients scheduled for coronary artery bypass surgery under oral therapy with 100 mg/d acetylsalicylic acid (aspirin). In each patient, three blood samples for platelet function analysis were taken before induction of anesthesia. The first sample was measured in the operating room (OR) area and designated as the reference value. The second sample was again measured by the same operator in the OR area using a random PTS transport. The third sample was sent to the central laboratory by PTS where it was measured by a local technician. Platelet function was assessed in whole blood by impedance aggregometry with a Multiplate™ analyzer using thrombin-related activation peptide (TRAP test) and arachidonic acid (ASPI test) (Dynabite GmbH, Munich, Germany). Mean ± SD for TRAP test was 1009 ± 196 in the reference measurement. Bias ± 95% limit of agreement between the reference measurement and a second measurement for TRAP test were 126 ± 284 (n = 25) for untransported and 181 ± 316 (n = 25) for PTS transported samples. In the reference measurements, 48/50 (96%) of TRAP values were within the normal range. After PTS transport, 35/50 (70%) of TRAP measurements in the central laboratory were within the normal range (p < 0.001). Mean ± SD for ASPI test was 175 ± 137. Bias ± 95% limit of agreement for ASPI test were 12 ± 109 (n = 25) for untransported and 68 ± 250 (n = 25) for PTS transported samples. In the reference measurements, 7/50 (14%) ASPI values were above the cut-off level and defined as reduced aspirin responsiveness. After PTS transport, only 1/50 (2%) of the patients showed reduced aspirin responsiveness in the central laboratory measurements (p = 0.031). In conclusion, PTS transport had a significant influence on platelet function testing by the Multiplate™ analyzer. Significantly fewer test results indicated normal platelet function in TRAP test and reduced aspirin responsiveness in ASPI test after PTS transport. Therefore, clinical decisions regarding platelet function and aspirin responsiveness should not be based on blood specimens transported by a PTS system.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Order of application and liver toxicity in patients given BU and CY containing conditioning regimens for allogeneic hematopoietic SCT

BU–CY is the established non-TBI-based myeloablative conditioning regimen for allogeneic hematopoietic SCT. However, liver toxicity and hepatic veno-occlusive disease (VOD) are frequent life-threatening complications. Pharmacological considerations suggest that BU can trigger toxicity of subsequent CY. Recent animal data confirmed this hypothesis. Less liver toxicity and better outcomes were observed when mice were treated with the reversed order of CY and BU. We analyzed in this study liver toxicity and outcome in patients receiving BU–CY (16 patients) or CY–BU (59 patients). Liver function differed significantly with higher levels of liver function tests between day +10 and +30, and a higher cumulative incidence of VOD in the BU–CY cohort (2/16 (12.5%) vs 0/59 (0%), P=0.006). TRM was significantly higher in patients receiving BU–CY (cumulative incidence BU–CY 45%, CY–BU 17%, P=0.02), without yet translating into a significant survival difference (incidence for survival: BU–CY 38%, CY–BU 63%; hazard ratio 1.19 for BU–CY, 95% confidence interval 0.29–4.82, P=0.80). Rates of engraftment and relapse were not different. These data support the concepts derived from animal models in favor of CY–BU compared with traditional BU–CY and form the basis for prospective controlled comparisons.