CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin alpha IIb beta 3.

UNLABELLED: Agonist-induced inside-out signaling activates platelet integrin alpha(IIb)beta(3), rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through alpha(IIb)beta(3) regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating alpha(IIb)beta(3) function. OBJECTIVE: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. METHODS AND RESULTS: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl(3)-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin beta(3) subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. CONCLUSION: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.

Characterization and chromosomal localization of JAM-1, a platelet receptor for a stimulatory monoclonal antibody.

We have previously reported the purification and characterization of a 32 kDa platelet surface glycoprotein that is recognized by the stimulatory monoclonal antibody, F11. The cDNA has been cloned and found to encode the human homolog of the murine junctional adhesion molecule, JAM; we therefore named this human homolog JAM-1. Northern blot analysis indicated that JAM-1 mRNA is expressed as multiple species, the predominant transcript being approximately 4.0 kb in size. Genetic mapping analysis using fluorescence in situ hybridization (FISH) showed that it is localized to chromosome 1q21.1-21.3. Recombinant JAM-1, when expressed in Chinese hamster ovary (CHO) cells, localized to the cell membrane with intense staining where two adjacent cells actually made contact with each other, suggesting that, similar to murine JAM, human JAM-1 may also localize at the cell-cell junction. In well-spread cells, JAM-1 co-localized with F-actin at the cell-cell contacts and at the membrane ruffles, but not at the stress fibers. Interestingly, JAM-1 localizes only to the cell-cell junctions formed by two transfected cells and not to the cell-cell junctions formed by a transfected cell with an untransfected cell, suggesting that JAM-1 may facilitate cell adhesion through homophilic binding. In addition, human platelets specifically bind to a monolayer of CHO cells expressing human JAM-1, further supporting homophilic interactions. The results presented here indicate that JAM-1, a receptor for a platelet-activating antibody, is the human homolog of the junctional adhesion molecule. JAM-1 is a single copy gene, which is constitutively expressed on various tissues and cells, and may be involved in cell to cell adhesion through homophilic interaction.

Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain.

Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity.

Developmental expression of the protein kinase C family in rat hippocampus.

Protein kinase C (PKC) is a heterogeneous family of ten or more isoforms which plays an important role in neuronal signal transduction. Isoforms from all subclasses are prominently expressed in the rat hippocampus, as demonstrated by immunoblot with isozyme-specific antisera: Ca(2+)-dependent (alpha, beta I, beta II and gamma), Ca(2+)-independent (delta, epsilon and a newly characterized PKC related to eta) and atypical (zeta). In addition, the zeta isoform is also found as the free, constitutively active catalytic domain, protein kinase M zeta (PKM zeta). Two distinct patterns of expression of PKC isozymes in rat hippocampus are found during development from E18 to P28. PKC zeta, PKM zeta and PKC delta are present at birth and their expression does not increase postnatally. In contrast, the other isoforms are expressed only at low levels at birth and then increase in the first 4 weeks postnatally. These two patterns of expression suggest distinct functions for PKC isozymes during development.

Persistent activation of the zeta isoform of protein kinase C in the maintenance of long-term potentiation.

Long-term potentiation in the CA1 region of the hippocampus, a model for memory formation in the brain, is divided into two phases. A transient process (induction) is initiated, which then generates a persistent mechanism (maintenance) for enhancing synaptic strength. Protein kinase C (PKC), a gene family of multiple isozymes, may play a role in both induction and maintenance. In region CA1 from rat hippocampal slices, most of the isozymes of PKC translocated to the particulate fraction 15 sec after a tetanus. The increase of PKC in the particulate fraction did not persist into the maintenance phase of long-term potentiation. In contrast, a constitutively active kinase, PKM, a form specific to a single isozyme (zeta), increased in the cytosol during the maintenance phase. The transition from translocation of PKC to formation of PKM may help to explain the molecular mechanisms of induction and maintenance of long-term potentiation.

Evidence for a new, high-molecular weight isoform of protein kinase C in rat hippocampus.

We describe a new form of protein kinase C (PKC) with a molecular weight of 97 kDa, higher than the known forms of vertebrate PKC. This putative new high-molecular weight isoform, which we are calling PKC (HMW), is increased in the membrane fraction either upon application of phorbol esters or with afferent synaptic stimulation of Schaffer collaterals in hippocampal slices. The protein cross-reacts on immunoblot with affinity-purified polyclonal antiserum raised against a peptide derived from the carboxy-terminus of PKC eta; it does not cross-react, however, with antiserum against the amino-terminal region of PKC eta. In the tissues examined, PKC(HMW) is localized primarily in brain, in contrast to PKC eta, which is found predominantly in lung and skin.