Arteriosclerosis, Thrombosis, and Vascular Biology, 2017
Experimental, clinical and pathological studies have indicated an important link between inflamma... more Experimental, clinical and pathological studies have indicated an important link between inflammation and thrombosis. However, the precise mechanisms regulating myeloid cell function and the importance of various myeloid lineages involved in thrombosis remain incompletely defined. Published work from our group shows that a systemic deficiency of KLF2 renders animals susceptible to thrombosis. Using conditional knockouts, we examined the role of myeloid KLF2 in thrombosis. Carotid thrombosis assay (Rose Bengal model) show a robust reduction in time to occlusion in MY-K2-KO mice. No difference was noted in complete blood counts and coagulation assays between MY-K2-KO and control mice. Adoptive transfer of KLF2-KO neutrophils significantly shortened the time to occlusive thrombosis in control mice compared with the occlusion time in control mice given control neutrophils. No change in thrombosis time was noted in MY-K2-KO mice transfused with either control or KLF2-KO neutrophils. Neut...
A 45 yo woman with a medical history of common variable immunodifficiency (CVID) was referred for... more A 45 yo woman with a medical history of common variable immunodifficiency (CVID) was referred for a bleeding disorder. The bleeding was manifested daily with ecchymosis and epistaxis, but prior to the visit, she developed a lower pelvic mass that was a massive hematoma requiring transfusion. In 2011 she had a splenectomy for thrombocytopenia. After splenectomy, her liver enlarged and one year prior to being seen, she was started on everolimus as part of a protocol to reduce her liver size. Bleeding started 3-6 months after starting everolimus treatment, first manifesting with recurrent lower gastrointestinal hemorrhage. In 2011 she had normal PT and aPTT. At the present time her PT remains normal at 11.43+0.67 (Mean+SD) (normal 9.7-12.7 sec); but her aPTT is abnormal at 43+6.1 (normal 28-38 sec); clottable fibrinogen (Fb) 305+72 (normal 200-400 mg/dl); Fb antigen, 428+115 mg/dl (normal 196-441); ratio of clottable Fb/Fb antigen = 0.71+ 0.06; reptilase time 20+2.2 (normal 14-23 sec);...
Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening emerg... more Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening emergency caused by auto-antibodies against ADAMTS13. Current standard therapy utilizes plasma exchange (PLEX) and corticosteroids. Although this induces remission, disease relapse remains a common problem. Since more than 90% of aTTP is caused by an antibody to ADAMTS13, additional immunosuppressive therapy with rituximab (RTX) given upfront has been shown to lower relapse rates. An alternative therapy, Cyclophosphamide (CTX) has been used in relapsed disease, but data on its use in upfront management is limited to case reports only. Our study aims to compare outcomes at our institution between upfront CTX, RTX, and standard therapy alone. Our secondary objective is to identify initial presenting features that are predictive of higher relapse risk. Methods: In a retrospective cohort study, we identified all patients at our institution with a diagnosis code of TTP (ICD 9, 10: 446.6, M31.1) be...
Experimental, clinical and pathological studies support an important link between inflammation an... more Experimental, clinical and pathological studies support an important link between inflammation and thrombosis. Although accumulating evidence suggests that cells of the innate immune system contribute to the thrombotic process, the identity of nodal molecular determinants operative in immune cells remains poorly understood. Our previous work in mice bearing global deletion of the transcription factor KLF2 identifiedthis factor as a critical mediator of thrombosis). Here, using cell-specific KLF2 deleted murine models (endothelial, platelet, and myeloid- deleted KLF2) we identify myeloid KLF2 as the major determinant of thrombosis. Complete blood counts and coagulation assays in myeloid KLF2 deleted mice (MY-K2-KO) were similar to those in control LysM cre mice. Using two models of vascular thrombosis (carotid artery thrombosis assay, Rose Bengal model and complete inferior vena cava ligation model of venous thrombosis) we observed a robust prothrombotic phenotype in the MY-K2-KO mic...
651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angio... more 651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype. Methods and Results. PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or pl...
Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatin... more Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatinib (Iclusig) is approved for CML patients with the T315I ABL kinase polymorphism. However, ponatinib treatment is associated with vascular events (myocardial infarction, stroke, coronary artery stenosis, limb ischemia and occlusion, and venous thrombosis) in~29% of patients. The mechanism(s) for these events has not been characterized. We developed a murine model to examine TKIs influence on arterial thrombosis risk. C57BL/6 mice, 18-22 weeks of age and treated with ponatinib by gavage for 14 days at 15 mg/kg PO BID, had significantly shorter carotid artery occlusion times induced by photochemical activation of Rose-Bengal compared to vehicle-treated mice (10.4 ± 2.9 min versus 32.3 ± 4.8 min, p < 0.0001). Mice were treated with ponatinib for 14 days at the 3 mg/kg PO BID, a dose that yields plasma concentrations similar to patients at 45 mg po daily, also had significantly shorter ve...
Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory respon... more Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis. Methods…
Previous studies have shown that the multiprotein kininogen (HK) and factor XII (FXII) receptor c... more Previous studies have shown that the multiprotein kininogen (HK) and factor XII (FXII) receptor complex on endothelial cells (HUVEC) that consists of gC1qR, uPAR, and cytokeratin 1 (Blood97:2342, 99:3585) allows for prekallikrein assembly and activation for bradykinin formation (Blood 103:4554). New studies show outside-in signaling through this HUVEC receptor complex. Single chain urokinase (ScuPA) or FXII in the presence of 0.05 mM zinc ion stimulates ERK1/2 (MAPK42 and 44) in HUVEC (Blood 106:Abstract 1024Blood 106:Abstract 2005). Furthermore, cleaved HK (HKa) or peptides from the HK domain 5 cell binding region block HUVEC ERK1/2 phosphorylation. The region on uPAR that mediates this signaling is the same 22 aa sequence on uPAR domain 2 that binds ScuPA, FXII, HK, and vitronectin (JBC 279:16621). ScuPA or FXII phosphorylation of ERK1/2 is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Additional investigations determined if ScuPA or FXII in the presence of zinc ...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Background: Terminal complications of bacterial sepsis include development of disseminated intrav... more Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-d...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Background: Terminal complications of bacterial sepsis include development of disseminated intrav... more Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-d...
SummaryThe plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent ... more SummaryThe plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on nonreduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of α-heli...
The Journal of laboratory and clinical medicine, 1988
High molecular weight kininogen (HMWK) is a multifunctional protein that is a parent molecule for... more High molecular weight kininogen (HMWK) is a multifunctional protein that is a parent molecule for bradykinin, a cofactor for coagulation, and an inhibitor of cysteine proteases. On immunoblot, nonreduced plasma HMWK is usually two bands at 140 kd and 120 kd; reduced plasma HMWK is a single band at 120 kd. In both concentration-dependent and time-dependent experiments kaolin-activated normal plasma HMWK becomes cleaved in an ordered sequence. When nonreduced, HMWK on immunoblot in kaolin-activated plasma changes in size from a 140 kd band through a 120 kd intermediate to result in a stable 100 kd protein. When reduced, HMWK on immunoblot in kaolin-activated plasma changes from a single 120 kd band through a 56 kd intermediate to result in a stable 46 kd protein. A similar sequence of cleavage of plasma HMWK occurs when the soluble activator dextran sulfate is used to stimulate the system. Cleavage of plasma HMWK after kaolin activation occurs similarly in factor XI-deficient plasma a...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2017
Experimental, clinical and pathological studies have indicated an important link between inflamma... more Experimental, clinical and pathological studies have indicated an important link between inflammation and thrombosis. However, the precise mechanisms regulating myeloid cell function and the importance of various myeloid lineages involved in thrombosis remain incompletely defined. Published work from our group shows that a systemic deficiency of KLF2 renders animals susceptible to thrombosis. Using conditional knockouts, we examined the role of myeloid KLF2 in thrombosis. Carotid thrombosis assay (Rose Bengal model) show a robust reduction in time to occlusion in MY-K2-KO mice. No difference was noted in complete blood counts and coagulation assays between MY-K2-KO and control mice. Adoptive transfer of KLF2-KO neutrophils significantly shortened the time to occlusive thrombosis in control mice compared with the occlusion time in control mice given control neutrophils. No change in thrombosis time was noted in MY-K2-KO mice transfused with either control or KLF2-KO neutrophils. Neut...
A 45 yo woman with a medical history of common variable immunodifficiency (CVID) was referred for... more A 45 yo woman with a medical history of common variable immunodifficiency (CVID) was referred for a bleeding disorder. The bleeding was manifested daily with ecchymosis and epistaxis, but prior to the visit, she developed a lower pelvic mass that was a massive hematoma requiring transfusion. In 2011 she had a splenectomy for thrombocytopenia. After splenectomy, her liver enlarged and one year prior to being seen, she was started on everolimus as part of a protocol to reduce her liver size. Bleeding started 3-6 months after starting everolimus treatment, first manifesting with recurrent lower gastrointestinal hemorrhage. In 2011 she had normal PT and aPTT. At the present time her PT remains normal at 11.43+0.67 (Mean+SD) (normal 9.7-12.7 sec); but her aPTT is abnormal at 43+6.1 (normal 28-38 sec); clottable fibrinogen (Fb) 305+72 (normal 200-400 mg/dl); Fb antigen, 428+115 mg/dl (normal 196-441); ratio of clottable Fb/Fb antigen = 0.71+ 0.06; reptilase time 20+2.2 (normal 14-23 sec);...
Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening emerg... more Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening emergency caused by auto-antibodies against ADAMTS13. Current standard therapy utilizes plasma exchange (PLEX) and corticosteroids. Although this induces remission, disease relapse remains a common problem. Since more than 90% of aTTP is caused by an antibody to ADAMTS13, additional immunosuppressive therapy with rituximab (RTX) given upfront has been shown to lower relapse rates. An alternative therapy, Cyclophosphamide (CTX) has been used in relapsed disease, but data on its use in upfront management is limited to case reports only. Our study aims to compare outcomes at our institution between upfront CTX, RTX, and standard therapy alone. Our secondary objective is to identify initial presenting features that are predictive of higher relapse risk. Methods: In a retrospective cohort study, we identified all patients at our institution with a diagnosis code of TTP (ICD 9, 10: 446.6, M31.1) be...
Experimental, clinical and pathological studies support an important link between inflammation an... more Experimental, clinical and pathological studies support an important link between inflammation and thrombosis. Although accumulating evidence suggests that cells of the innate immune system contribute to the thrombotic process, the identity of nodal molecular determinants operative in immune cells remains poorly understood. Our previous work in mice bearing global deletion of the transcription factor KLF2 identifiedthis factor as a critical mediator of thrombosis). Here, using cell-specific KLF2 deleted murine models (endothelial, platelet, and myeloid- deleted KLF2) we identify myeloid KLF2 as the major determinant of thrombosis. Complete blood counts and coagulation assays in myeloid KLF2 deleted mice (MY-K2-KO) were similar to those in control LysM cre mice. Using two models of vascular thrombosis (carotid artery thrombosis assay, Rose Bengal model and complete inferior vena cava ligation model of venous thrombosis) we observed a robust prothrombotic phenotype in the MY-K2-KO mic...
651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angio... more 651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype. Methods and Results. PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or pl...
Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatin... more Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatinib (Iclusig) is approved for CML patients with the T315I ABL kinase polymorphism. However, ponatinib treatment is associated with vascular events (myocardial infarction, stroke, coronary artery stenosis, limb ischemia and occlusion, and venous thrombosis) in~29% of patients. The mechanism(s) for these events has not been characterized. We developed a murine model to examine TKIs influence on arterial thrombosis risk. C57BL/6 mice, 18-22 weeks of age and treated with ponatinib by gavage for 14 days at 15 mg/kg PO BID, had significantly shorter carotid artery occlusion times induced by photochemical activation of Rose-Bengal compared to vehicle-treated mice (10.4 ± 2.9 min versus 32.3 ± 4.8 min, p < 0.0001). Mice were treated with ponatinib for 14 days at the 3 mg/kg PO BID, a dose that yields plasma concentrations similar to patients at 45 mg po daily, also had significantly shorter ve...
Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory respon... more Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis. Methods…
Previous studies have shown that the multiprotein kininogen (HK) and factor XII (FXII) receptor c... more Previous studies have shown that the multiprotein kininogen (HK) and factor XII (FXII) receptor complex on endothelial cells (HUVEC) that consists of gC1qR, uPAR, and cytokeratin 1 (Blood97:2342, 99:3585) allows for prekallikrein assembly and activation for bradykinin formation (Blood 103:4554). New studies show outside-in signaling through this HUVEC receptor complex. Single chain urokinase (ScuPA) or FXII in the presence of 0.05 mM zinc ion stimulates ERK1/2 (MAPK42 and 44) in HUVEC (Blood 106:Abstract 1024Blood 106:Abstract 2005). Furthermore, cleaved HK (HKa) or peptides from the HK domain 5 cell binding region block HUVEC ERK1/2 phosphorylation. The region on uPAR that mediates this signaling is the same 22 aa sequence on uPAR domain 2 that binds ScuPA, FXII, HK, and vitronectin (JBC 279:16621). ScuPA or FXII phosphorylation of ERK1/2 is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Additional investigations determined if ScuPA or FXII in the presence of zinc ...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Background: Terminal complications of bacterial sepsis include development of disseminated intrav... more Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-d...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Background: Terminal complications of bacterial sepsis include development of disseminated intrav... more Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-d...
SummaryThe plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent ... more SummaryThe plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on nonreduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of α-heli...
The Journal of laboratory and clinical medicine, 1988
High molecular weight kininogen (HMWK) is a multifunctional protein that is a parent molecule for... more High molecular weight kininogen (HMWK) is a multifunctional protein that is a parent molecule for bradykinin, a cofactor for coagulation, and an inhibitor of cysteine proteases. On immunoblot, nonreduced plasma HMWK is usually two bands at 140 kd and 120 kd; reduced plasma HMWK is a single band at 120 kd. In both concentration-dependent and time-dependent experiments kaolin-activated normal plasma HMWK becomes cleaved in an ordered sequence. When nonreduced, HMWK on immunoblot in kaolin-activated plasma changes in size from a 140 kd band through a 120 kd intermediate to result in a stable 100 kd protein. When reduced, HMWK on immunoblot in kaolin-activated plasma changes from a single 120 kd band through a 56 kd intermediate to result in a stable 46 kd protein. A similar sequence of cleavage of plasma HMWK occurs when the soluble activator dextran sulfate is used to stimulate the system. Cleavage of plasma HMWK after kaolin activation occurs similarly in factor XI-deficient plasma a...
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Papers by Alvin Schmaier