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The Calibrated Automated Thrombogram (CAT), a plate-based assay that measures thrombin generation and inhibition in plasma samples, is modified to measure the procoagulant activity of phospholipid associated with plasma microparticles (MP). The assay uses a tissue factor trigger without addition of 4 μM exogenous phospholipid (PL) used in the standard CAT. Calibrated with 4:1 posphatidylcholine- phosphatidylserine (PCPS) liposomes, the assay defines a median of 40 nM procoagulant phospholipid (PPL) equivalents in plasma containing MPs from 50 normal donors, with a range from 20 - 200 nM. Like the standard CAT, the modified assay detected no difference in plasma from 36 individuals with a history of a single episode of venous thromboembolism. However the male cases had double the PPL activity, as measured by rate of thrombin generation, of females; and there was a significant correlation among cases of increased thrombin generation with age. In contrast, there were no gender disparities or age correlations among control plasmas. The findings suggest that procoagulant activity of plasma microparticles, facilitated by a simplified, one-stage plate-based assay, offer a promising avenue of investigation of mechanisms and management of venous thromboembolic disorders., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Introduction: Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps., Materials/methods: VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed., Results: The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas., Conclusions: The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Post-menopausal hormone therapy increases the risk for venous thrombosis, and possibly myocardial infarction (MI) and ischemic stroke. However, most women using hormone therapy do not suffer thrombosis, and to date our ability to identify women at risk is limited. Thrombosis, arterial or venous, has 2 requisites: a vascular anomaly and a response of the hemostasis system to the anomaly. Consequently, experimental approaches to understand the pathophysiology of thrombosis require definition of vascular anatomy and function as well as characteristics of the blood within the context of genetic background, lifestyle choices and environmental exposures, which influence gene expression. Defining interactions among factors that affect individual propensity to thrombosis will allow physicians to better identify at-risk individuals, for example a woman contemplating estrogen therapy for symptoms of menopause, and prevent adverse thrombotic events.

Introduction: To test the hypothesis that circulating platelets display evidence of reversible interactions with atherosclerotic lesions, platelet alpha-granule content and propensity for microaggregate formation were measured in samples from normal donors (n=65) and from patients with either peripheral arterial disease (n=47) or renovascular hypertension (n=22). To measure the effect of a defined arterial injury on platelet function, platelet samples were compared before and 30 min after elective angioplasty., Materials and Methods: P-selectin was measured after strong stimulation of ultra-dilute platelets with thrombin (10 nM). Microaggregation was measured as a platelet count deficit in citrate-anticoagulated platelet-rich plasma (PRP) relative to that predicted from the count in EDTA-anticoagulated blood., Results: Platelet alpha-granule P-selectin was significantly lower from platelets of patients compared to normal donors. In addition, platelets from patients have a significantly greater propensity to form microaggregates in citrate anticoagulant. In contrast to atherosclerotic renovascular hypertension, platelets from patients with fibromuscular dysplasia, a distinct non-inflammatory cause of arterial stenosis, do not differ significantly from normal donors. Other than the PRP platelet count, which rose transiently following angioplasty, other platelet measures were unchanged by the injury., Conclusions: Atherosclerotic arterial disease is associated with an increased share of platelets unable to express P-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. These platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process.

A simple, sensitive and specific assay for plasminogen activators is described. The assay utilizes fluorescein-labeled fibrinogen or fibrin at low concentrations, and enables simultaneous evaluation of the plasminogen and fibrin dependence of the reaction, that is, discrimination of tissue-type and urokinase-type plasminogen activators, and non-specific proteolysis. Addition of antisera verify identification of the activator species. The assay reagent contains plasminogen and fluorescein-labeled fibrinogen, to which is added the specimen and then then thrombin, either at the initiation or the termination of the reaction. Supernatant fluorescence is proportional to plasminogen activator concentration. With a four-hour incubation, 1 milliunit (14 pg) of tissue (melanoma) plasminogen activator (TPA) or 2 milliunit (36 pg) of urokinase (UK) may be detected.