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Biomedical subjects

Michael H Kroll

Publications and source records attributed to Michael H Kroll.

12 recordsLinked to original sources
PubMed2006

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling.

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its beta-subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin alpha(IIb)beta(3) is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on alpha(IIb)beta(3), we examined alpha(IIb)beta(3)/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that alpha-actinin, myosin heavy chain, and Syk coimmunoprecipitate with alpha(IIb)beta(3) in resting platelets and that 120 dyn/cm(2) shear stress leads to their disassociation from alpha(IIb)beta(3). Shear-induced disassociation of alpha-actinin and myosin heavy chain from the beta(3) tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to alpha(IIb)beta(3). Syk's disassociation from beta(3) is inhibited when VWF binding to either GpIb-IX-V or alpha(IIb)beta(3) is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to alpha(IIb)beta(3) but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing alpha(IIb)beta(3) with beta(3) truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates alpha(IIb)beta(3) function and suggest that shear-induced alpha(IIb)beta(3)-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the beta(3)-tail.

Actinin↗
PubMed2005

Acquired disorders of platelet function.

A qualitative abnormality of platelet function should be considered in patients with mucocutaneous bleeding in the absence of thrombocytopenia or von Willebrand disease. Antiplatelet drugs are the most common cause of acquired platelet disorders leading to bleeding. Uremia, hepatic cirrhosis, myeloma and related disorders, polycythemia vera, essential thrombocythemia, and cardiopulmonary bypass have long been recognized as clinical situations in which platelet dysfunction may contribute to bleeding. When an acquired platelet disorder is suspected, it is useful to examine platelet function by measuring the bleeding time, examining platelet-dependent closure time in a platelet function analyzer and performing platelet aggregometry. When a specific acquired platelet disorder is diagnosed, many treatment options are available including controlling the underlying disease, giving platelet transfusions and administering a hemostatic drug.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Targeting shear stress-induced platelet activation: is lesion-specific antiplatelet therapy a realistic clinical goal?

Platelets are mediators of physiologic hemostasis and pathologic thrombosis. They operate within distinctive vascular and rheologic microenvironments, and their participation in hemostasis or thrombosis is directed by distinct variables operating within the microenvironment. Thrombosis is not simply too much hemostasis: there is good evidence that triggering mechanisms of platelet aggregation under low shear stress conditions are different from those operating under high shear stress conditions. Such differences are hypothesized to exist in vivo and to separate mechanisms of microvascular hemostasis from mechanisms of arterial thrombosis, such as those involved in myocardial, cerebral and peripheral vascular ischemia and infarction. This separation forms the conceptual basis for the hypothesis that lesion-specific antithrombotic agents might some day be invented that inhibit arterial thrombosis without causing bleeding that arises from impaired hemostasis. The focus of much of the work in this field has been platelet aggregation initiated by shear-dependent von Willebrand factor binding to the platelet glycoprotein Ib-IX-V complex. It is hypothesized that by elucidating molecular mechanisms of platelet activation operating under pathologically elevated shear stresses, targets of lesion-specific therapies will one day be identified for use in clinical syndromes of arterial thrombosis.

Animals↗
PubMed2004

Filamin A binding stabilizes nascent glycoprotein Ibalpha trafficking and thereby enhances its surface expression.

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.

Acetylcysteine↗
PubMed2004

Role of the Pyk2-MAP kinase-cPLA2 signaling pathway in shear-dependent platelet aggregation.

Mechanisms of shear-induced platelet aggregation are not established. Data that ristocetin-induced von Willebrand factor (VWF) binding to glycoprotein (Gp) Ibalpha activates proline-rich tyrosine kinase 2 (Pyk2) and extracellular-regulated kinase (ERK) has led to speculation that these events are coupled and that a MAP kinase may activate cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release. To test this hypothesis and clarify the role of AA metabolism in shear-induced VWF-dependent platelet aggregation, we examined Pyk2, ERK1/2, and p38 phosphorylation, and arachidonic acid release and metabolism in platelets subjected to pathological shear stress in vitro. We observe tyrosine phosphorylation of Pyk2, p38, and ERK1/2 but no measurable increase in free AA, 12-hydroxyeicosatetraenoic acid, or thromboxane A2. Inhibitors of ERK, p38, or cyclooxygenase activation fail to affect shear-induced platelet aggregation. When washed platelets are aspirin-pretreated, arachidonic acid release becomes measurable and aggregation at 60 and 120 s is attenuated. These data indicate that shear-induced VWF binding to platelet GpIb-IX-V activates Pyk2, ERK1/2, p38, and cPLA2, but that the magnitude of these responses is below the threshold needed to enhance shear-induced VWF-dependent platelet aggregation in the presence of plasma. These results provide a mechanistic basis for the long-standing observation that shear-dependent platelet aggregation is unaffected by the antiplatelet drug aspirin.

Acetyltransferases↗
PubMed2003

Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation.

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.

Amino Acid Sequence↗
PubMed2003

Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation.

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm(2) (shear rate of 6,000 sec(-1)). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP(3) production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP(3) production and aggregation. Selective blockade of the P2Y(12) receptor results in the inhibition of Syk phosphorylation, PIP(3) production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y(12) receptor-mediated activation of Syk.

Blood Platelets↗
PubMed2002

AIDS-related systemic non-Hodgkin's lymphoma at a large community program.

The introduction of triple antiretroviral therapy has led to reductions in opportunistic diseases in HIV-infected patients. However, little is known of the effect of this therapy on the clinical and pathological features and the outcome of patients with AIDS-related systemic non-Hodgkin's lymphoma (NHL). We examined the incidence and clinical manifestations of HIV-infected patients with systemic NHL at the Harris County Hospital District and Veterans Affairs Medical Center (Houston, TX). Between January 1996 and December 2000, 76 cases of systemic NHL were diagnosed in 3655 HIV-infected patients. Three groups of patients diagnosed with systemic NHL were identified according to their history of antiretroviral therapy: treatment naive (n = 20), dual nucleoside (n = 22), and triple antiretroviral drug-treated patients (n = 34). The median duration of antiretroviral therapy before the diagnosis of systemic NHL in the triple antiretroviral and dual nucleoside treatment groups was 12 versus 8 months (p < 0.0004). Thirty-five percent of patients (12 of 34) in the triple treatment group had an HIV RNA viral load of <400 copies/ml and their median CD4+ cell count was 301 cells/mm(3) (range, 46 to 667 cells/mm(3)) at the time of diagnosis of systemic NHL. More patients treated with triple antiretroviral therapy received complete courses of chemotherapy as compared with the other two groups (p = 0.013). However, the overall survival did not differ significantly among the three groups of patients. These data suggest that AIDS-related systemic NHL continues to occur even in patients treated with triple antiretroviral therapy. In addition, this opportunistic malignancy is associated with significant mortality. Therefore, it is necessary to develop a better understanding of the pathogenesis of this disease.

Adult↗
PubMed2002

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.

Actinin↗
PubMed2002

Relapse of thrombotic thrombocytopenic purpura associated with decreased VWF cleaving activity.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon syndrome characterized by reversible, systemic aggregation of platelets in the microcirculation and disseminated microvascular thrombosis. Surgery may precipitate TTP and has been associated with relapse in some patients. However, relapse of this life-threatening disorder is unpredictable. We report a patient with an antecedent history of TTP who experienced a relapse after elective cardiac surgery. In this case, decreased von Willebrand factor (vWF)-cleaving metalloproteinase activity and an inhibitor of this endogenous enzyme were demonstrated preoperatively. These findings suggest that decreased vWF-cleaving metalloproteinase activity and/or the presence of its inhibitor may predict an increased risk for surgical-associated relapse of TTP.

Adult↗