IntroductionPlatelets adhere at sites of vascular injury to prevent hemorrhage 1 but may also form occluding arterial thrombi that cause disease. 2,3 In vessels in which rapid blood flow creates high wall-shear rates, such as arterioles in the normal circulation or atherosclerotic arteries with restricted lumen, platelet thrombus formation depends on von Willebrand factor (VWF) immobilized on extracellular matrix components, particularly collagens. 4 Binding of glycoprotein Ib␣ (GPIb␣), a constituent of the GPIb-IX-V complex, to the VWF A1 domain (A1VWF) initiates platelet tethering to these surfaces but by itself can only support translocation with stopand-go motion. 5 Once tethered, however, platelets rapidly achieve irreversible adhesion mediated by different integrins, including ␣ IIb ␤ 3 bound to the Arg-Gly-Asp (RGD) motif in the VWF C1 domain. 4,5 Activated ␣ IIb ␤ 3 serves also to immobilize on the surface of adherent platelets the plasma proteins, mainly VWF and fibrinogen, that mediate the recruitment of additional platelets into the forming thrombus. 6 Platelet activation, necessary to promote the ligand-binding function of ␣ IIb ␤ 3 , is coupled to the interactions that establish initial platelet-surface contacts, as shown by the fact that VWF binding to GPIb␣ leads to aggregation. 7,8 Sustained elevations of intracellular calcium concentration ([Ca ϩϩ ] i ), a marker of activation, occur in association with shear-induced platelet aggregation dependent on VWF and GPIb␣ 9 and may be the consequence of a transmembrane ion flux. 10 Oscillations of [Ca ϩϩ ] i have also been observed to accompany platelet adhesion to VWF, but this finding has been given discordant interpretations. 11,12 Some results 13 suggest that the VWF-GPIb␣ interaction may induce transient elevations (spikes) of [Ca ϩϩ ] i that activate ␣ IIb ␤ 3 in an initially reversible manner and influence the dynamic aspect of platelet-surface contacts before stable adhesion is established. This is in contrast to the idea that transient tethering to immobilized VWF depends only on GPIb␣, whereas activation of ␣ IIb ␤ 3 , like that of other integrins on leukocytes, 14 leads to irreversible adhesion. 4,5 Moreover, it has been proposed that phosphatidylinositol 3-kinase (PI3-K) plays an essential role in the activation of ␣ IIb ␤ 3 required for stable platelet adhesion. 15 To evaluate these conclusions, we concurrently analyzed the instantaneous velocity and [Ca ϩϩ ] i in single platelets interacting with immobilized VWF. We identified a sequence of distinct cytosolic Ca ϩϩ elevations associated with a 2-step process For personal use only. on May 10, 2018. by guest www.bloodjournal.org From of ␣ IIb ␤ 3 activation. The first signal involves release from intracellular stores and always precedes stationary adhesion. The second signal, which is coupled to adenosine diphosphate (ADP)-receptor stimulation and is inhibited by wortmannin, follows stationary adhesion but precedes the initiation of platelet aggregation on the surface. These results challeng...

IntroductionThe initial adhesion of platelets to the extracellular matrix of injured blood vessels is mediated at high shear rates by von Willebrand factor (VWF) interaction with glycoprotein (GP) Ib-IX-V. 1 Additionally, engagement of GP Ib-IX-V by VWF is thought to contribute to stable platelet adhesion by generating intracellular signals necessary for activation of ␣IIb␤3. Indeed, ␣IIb␤3 activation and platelet thrombus formation have been observed under a number of experimental conditions following platelet interaction with VWF. [2][3][4][5][6] Furthermore, specific biochemical responses have been documented under the same conditions, including induction of Ca 2ϩ fluxes and activation of tyrosine, serine-threonine, and lipid kinases. 7,8 Consequently, GP Ib-IX-V may function as a signaling receptor and an adhesion receptor.GP Ib-IX-V is a complex of 4 transmembrane polypeptides. 9-11 Although the cytoplasmic tail of each subunit lacks a catalytic domain, each may interact directly or indirectly with proteins that can transmit intracellular signals. For example, the cytoplasmic tail of GP Ib␣ can interact directly with filamin, GP Ib␣ and Ib␤ with 14-3-3-, and GP Ib␤ and GP V with calmodulin. 12-15 GP Ib-IX-V can be coimmunoprecipitated from platelets with signaling molecules, including Src family kinases, 16 phosphatidylinositol 3-kinase (PI 3-kinase) 17 and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2). 18 Furthermore, VWF-dependent platelet activation may require localization of GP Ib-IX-V to lipid rafts, membrane structures implicated in cellular signaling. 19 Although there is good evidence for a functional link between GP Ib-IX-V and ␣IIb␤3, 2 critical questions remain: Is GP Ib-IX-V itself capable of transducing signals in platelets, and, if so, to what extent do these signals participate in the activation of ␣IIb␤3? Several factors have conspired to make it difficult to answer these questions. First, a subpopulation of GP Ib-IX-V in platelets may be associated with immunoreceptor tyrosine activation motif (ITAM)-bearing proteins that can signal in their own right, including the Fc␥RIIA receptor and the FcR ␥-chain. 20,21 Second, platelets express numerous receptors for soluble and matrix-associated agonists, and some of the agonists (eg, adenosine diphosphate [ADP], thromboxane A 2 ) are released by adherent platelets. 22 Third, VWF not only interacts with GP Ib-IX-V through its A1 domain but also with ␣IIb␤3 through its C1 domain. 1 Thus, outside-in signals stimulated by VWF binding to ␣IIb␤3 can confound analysis of GP Ib-IX-V signaling. 23 Finally, studies of GP Ib-IX-V signaling under static conditions have frequently utilized nonphysiological mediators, such as botrocetin or ristocetin, to promote VWF binding to GP Ib-IX-V, complicating data interpretation further.Thus, despite the publication of many important studies on the molecular contributors to signaling responses downstream of GP Ib-IX-V, most to date have failed to consistently employ conditions to avoi...

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Published studies, taken together, suggest the existence of a single canine lymphoma entity, with a small clear cell appearance by cytological evaluation, a histopathological T-zone pattern and an aberrant CD45-negative T-cell phenotype, mostly characterized by long-term survival. We describe clinical presentation and outcome in a retrospective case series of canine small clear cell/T-zone lymphoma. Despite the reported predisposition of Golden retriever, this breed was not represented in our case series. Most dogs presented with stage V disease, whereas only few had clinical signs or peripheral cytopenias. Blood was almost always more infiltrated than bone marrow. Median survival confirmed the favourable prognosis described in literature, but a few dogs died within a short time. Also, a subgroup of dogs developed second malignancies, eventually leading to death. We did not investigate possible prognostic factors because of the wide variety in treatments, and further studies are needed to identify high-risk animals.

We have investigated the role of adenosine diphosphate (ADP) receptors in the adhesion, activation, and aggregation of platelets perfused over immobilized von Willebrand factor (VWF) under high shear stress. Blocking P2Y(1) prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of alpha(IIb)beta(3) activation, and decreased the duration of transient arrests from 5.9 seconds +/- 2.8 seconds to 1.2 seconds +/- 0.8 seconds; in contrast, blocking P2Y(12) inhibited only the formation of larger aggregates. Moreover, blocking P2Y(1) decreased the proportion of platelets showing early intracytoplasmic Ca(++) elevations (alpha/beta peaks) from 20.6% +/- 1.6% to 14.6% +/- 1.5% (P< .01), and the corresponding peak ion concentration from 1543 nM +/- 312 nM to 1037 nM +/- 322 nM (P < .05); it also abolished the Ca(++) elevations seen in firmly attached platelets (gamma peaks). Blocking P2Y(12) had no effect on these parameters, and did not enhance the effect of inhibiting P2Y(1). Inhibition of phospholipase C had similar consequences as the blocking of P2Y(1), whereas inhibition of Src family kinases abolished both type alpha/beta and gamma Ca(++) oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y(1) may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y(12) to sustained thrombus formation.

Objective-Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury.FN containing Extra Domain A (EDA ϩ FN) is absent from normal plasma, but elevated plasma levels of EDA ϩ FN are found in several pathological conditions. We hypothesized that EDA ϩ FN plays a special role in thrombosis. Methods and Results-We used mouse strains constitutively including (EDA ϩ/ϩ ) or excluding (EDA Ϫ/Ϫ ) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA ϩ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA ϩ/ϩ mice thrombi (Ͼ30 m) grew faster when compared with EDA WT/WT (6.6Ϯ0.2 minutes versus 8.3Ϯ0.6 minutes, PϽ0.05) and the mean vessel occlusion time was shorter (9.9Ϯ0.4 minutes versus 14.6Ϯ1.7 minutes, PϽ0.05). However, the presence of EDA ϩ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA ϩ/ϩ mice after collagen/epinephrine infusion was twice that of EDA WT/WT or EDA Ϫ/Ϫ mice. Conclusions-Our findings reveal that EDAϩ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated. (Arterioscler Thromb Vasc Biol. 2008;28:296-301) Key Words: plasma fibronectin Ⅲ fibronectin splice variants isoforms Ⅲ arterial thrombosis Ⅲ intravital microscopy Ⅲ thromboembolism Ⅲ arterial and venous injury T he formation of a thrombus in an injured vessel wall is a complex process that involves multiple adhesion molecules and their respective receptors on the platelet surface. von Willebrand factor (vWF) and fibrinogen are considered the major ligands mediating platelet adhesion and aggregation. However, in an experimental model 73% of the injured vessels of mice lacking both fibrinogen and vWF still formed occlusive thrombi either at the site of injury or downstream, 1 suggesting that other major adhesive proteins, such as fibronectin (FN), might contribute to the process. Incorporation of FN into a growing thrombus was shown both in vitro 2 and in vivo. 3 Additionally, it was documented in vivo that the depletion or lower levels (50%) of plasma FN result in serious defects in arterial thrombosis. 3,4 FN is a dimeric multidomain glycoprotein playing an important role in adhesion, migration, growth and differentiation of cells. 5,6 FN generates protein diversity as a consequence of alternative processing of a single primary transcript at 3 sites: the Extra Domain B (EDB, EDII, or EIII-B), Extra Domain A (EDA, EDI, or EIII-A), and the Type III Homologies Connecting Segment (IIICS) ( Figure 1A). 7-9 Two major forms of FN exist: soluble plasma FN (pFN), which lacks both the EDA and EDB domains ( Figure 1A); and cellular FN (cFN), which is deposited as insoluble fibrils in the extracellular matrix (ECM) and contains these domains at variable proportions.FN is a ligand for many members of the integrin receptor family and binds to thrombosis-related protein...

Canine acute leukaemias (ALs) have a poor prognosis, with reported survival times (ST) of only a few weeks or months. Also, clinical studies assessing prognostic factors are lacking. This study aims to retrospectively assess variables that predict ST in dogs with AL, and to identify correlations between outcome and therapeutic protocols. Diagnosis and sub-classification into AL subtypes was made based on haematological findings, morphological assessment and flow cytometric immunophenotyping. Clinical-pathological features of AL subtypes at presentation concurred with those described in the literature. A normal neutrophil count at presentation significantly prolonged ST (P = 0.027). Additionally, there was a trend for anaemic dogs to have shorter survival compared with those without anaemia, and the incorporation of cytosine in the chemotherapy protocol produced a moderate but not significant increase in median ST for dogs with AL. Further prospective studies with standardized treatments are needed to confirm and improve our results.

Primary effusion lymphoma (PEL) is a peculiar B-cell lymphoma characterized by infection by human herpesvirus type-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV) and by preferential growth in the serous body cavities. Histogenetic studies have suggested that PEL originates from B cells at a late stage of differentiation. In this study, we have investigated PEL for the expression status of MUM1/IRF4 (multiple myeloma 1/interferon regulatory factor 4) protein, which is involved in physiological B-cell maturation and represents a histogenetic marker of late B-cell differentiation. Using multiple detection assays, all cases of PEL (n = 22) were found to express MUM1/IRF4 molecules. MUM1/IRF4 expression was a selective feature of PEL among lymphomas involving the serous body cavities as secondary lymphomatous effusions generally failed to express the protein. In reactive lymphoid tissues, MUM1/ IRF4 expression clustered with advanced stages of B-cell differentiation. Comparison of MUM1/IRF4 expression with that of other histogenetic markers defined two phenotypic variants of PEL, i.e. MUM1/IRF4+, CD138/syndecan-1+, B-cell antigen- (20 out of 22 cases) and MUM1/IRF4+, CD138/syndecan-1-, B-cell antigen+ (2 out of 22 cases), suggesting a certain degree of heterogeneity in the disease histogenesis. The implications of these data are threefold. First, MUM1/IRF4 expression corroborates the notion that PEL originates from post-germinal centre, preterminally differentiated B-cells. Second, MUM1/IRF4 may help in the differential diagnosis of PEL among other lymphomas involving the serous body cavities. Finally, MUM1/IRF4 may interact with HHV-8/KSHV-encoded interferon regulatory factors (IRFs) and thus contribute to PEL escape from interferon-mediated control of viral infection.