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This study evaluated the role of circulating tissue factor (TF) in mediating thrombus formation on stents in an in vitro model of stent perfusion.The traditional view of coagulation has recently been challenged by the demonstration that TF is present in circulating blood. The potential contribution of this intravascular pool of TF to thrombus formation on stents is not known.Coronary stents were placed in parallel silicone tubes connected to a roller pump that was set to pump blood at a flow rate of 10 ml/min. Stents were then exposed to heparinized blood from healthy volunteers for 120 min.The presence of the stent in the circuit caused a significant increase in monocyte TF expression, but only monocytes with attached platelets stained positive for TF. Thrombi formed on stents and the thrombi stained positive for TF. Pretreatment of blood with a monoclonal antibody against TF (cH36) caused a 56% reduction in 125I-fibrin(ogen) deposition on stents compared with controls (p = 0.002). Monocyte depletion of blood reduced 125I-fibrin(ogen) deposition by 45% (p = 0.01) and TF staining in the thrombus by 83% (p = 0.01). Pretreatment of blood with a monoclonal antibody against P-selectin reduced 125I-fibrin(ogen) deposition by 24% (p = 0.04). Perfusion of stents with leukocyte-reduced platelet-rich plasma (PRP) produced small thrombi and treatment of PRP with cH36 reduced 125I-fibrin(ogen) deposition by 43% (p = 0.01).Circulating TF plays a pivotal role in thrombus formation on stents. Monocytes appear to be the main, but not only, source of TF depositing in the thrombus. [Copyright &y& Elsevier]

Glycoprotein (GP) VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiological collagen receptor from studies of patients deficient in this protein. GPVI-deficient platelets lacked specifically collagen-induced aggregation and the ability to form thrombi on a collagen surface under flow conditions, suggesting that GPVI makes an indispensable contribution to collagen-induced platelet activation. On the platelet surface, GPVI is present as a complex with the Fc receptor (FcR) γ-chain, probably composed of two GPVI molecules and one FcR γ-chain dimer. GPVI must form such a dimeric complex to exhibit high affinity binding to collagen. The GPVI-induced activation mechanism is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR γ-chain, and then this signal is transduced to many related proteins, mainly by tyrosine phosphorylation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow. However, individuals with GPVI-deficient or null platelets do not exhibit any strong bleeding tendency. Analyzing this apparent dichotomy should provide us with a more precise understanding of the mechanism of thrombus formation. [Copyright &y& Elsevier]

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with α2β1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of α2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains. [Copyright &y& Elsevier]

Cyclic prodrug 1 was derived from RGD peptidomimetic 2 by linking the amino and carboxylic acid groups via an (acyloxy)alkoxy linker. The formation of a cyclic prodrug can transiently alter the physicochemical properties of the RGD peptidomimetic. Cyclic prodrug 1 was synthesized via the key intermediate 8, and the synthesis of this key intermediate was accomplished using two different routes. Cyclic prodrug 1 has a smaller hydrodynamic radius and higher membrane interaction potential than those of the parent RGD peptidomimetic 2. The cell membrane permeation of cyclic prodrug 1 is twice that of the parent peptidomimetic 2. The prodrug-to-drug conversion can be carried out in isolated porcine esterase as well as human plasma. The cyclic prodrug is more stable at pH 4 than pH 7, and is very unstable at pH 10. The cyclic prodrug has antithrombic activity, suggesting that it can be converted to the RGD peptidomimetic in platelet-rich plasma (PRP). [Copyright &y& Elsevier]

A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100×g for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71±0.30%; n=15) suspension of highly viable (98.72±0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89±1.59%; n=9) and higher yields (56.56±3.89%, n=9 versus 36.11±2.23%, n=15). Where needed, a further centrifugation at 250×g for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n=15) contaminating platelets. A suspension of 85.96±2.20% pure granulocytes (93.23±1.74% neutrophils; n=14) with minimal contamination by erythrocytes and high viability (93.11±1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible. [Copyright &y& Elsevier]

A new in vitro model is proposed for studying the spatiotemporal distributions of activated clotting factors, in which clotting is activated in a thin layer of nonstirred plasma supplemented with a fluorogenic substrate and is monitored by fluorescence from its cleavage product. Analysis of the spatiotemporal dynamics of factor XIa and kallikrein in glass-activated human plasma provides evidence that both contact factors remain restricted to the glass surface and possibly a narrow boundary zone (<0.1 mm). The kinetics of factor XIa and kallikrein studied by a new method (in nonstirred plasma) coincided with those studied fluorimetrically with full stirring: their concentrations rapidly rose for the first few minutes after activation and then slowly declined. Factor XI and prekallikrein activation is likely to be restricted by the limited number of sites available for binding to the surface. The maximum concentration of the active factors was estimated at 2×108 molecules per mm2 at the glass surface (irrespective of stirring). At the plastic surface, this value was 15–30 times lower. [Copyright &y& Elsevier]