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Papers by Canio Refino

Thrombosis and Haemostasis

SummaryClinical experience suggests that thrombolytic-induced bleeding is associated with systemi... more SummaryClinical experience suggests that thrombolytic-induced bleeding is associated with systemic activation of the thrombolytic system. Using fibrin specific variants of tissue-type plasminogen activator (t-PA) and making use of the apparent fibrin specificity of streptokinase (SK) in the rabbit we tested the hypothesis that minimizing systemic plasmin production and fibrinogenolysis will decrease hemorrhages in models of peripheral bleeding and embolic stroke. t-PA consumed 51% of the available fibrinogen; caused cerebral bleeds and increased peripheral bleeding time. Fibrin-specific variants of t-PA depleted less than 20% of the fibrinogen and did not cause peripheral or cerebral bleeding. However, an equipotent dose of SK converted only 12% of the available fibrinogen but increased bleeding time and caused hemorrhagic conversion in 75% of embolic stroke model animals treated. The data suggest that bleeding associated with tissue-type plasminogen activators is linked to systemic...

Thrombosis and Haemostasis

Summary10C12, a human antibody F(ab’)2, which specifically binds to the Gla domain of factor IX, ... more Summary10C12, a human antibody F(ab’)2, which specifically binds to the Gla domain of factor IX, interfered with all known coagulation processes that involve factor IX/IXa. These include the function of the intrinsic Xase complex and the activation of zymogen factor IX by factor XIa and by the tissue factor:factor VIIa complex. Furthermore, 10C12 potently inhibited activated partial thromboplastin clotting times (APTT) in plasma of guinea pig and rat, thus enabling in-vivo evaluation. In guinea pigs, a bolus administration of 10C12 (10 μg/kg) prevented cyclic flow variations in damaged carotid arteries without affecting coagulation or bleeding parameters. At a 100-fold higher dose, 10C12 had no effect on normal hemostasis as assessed by the cuticle bleeding time. At this dose, 10C12 was also efficacious in a rat arterial thrombosis model, substantially reducing clot weight and duration of vessel occlusion while prolonging ex-vivo APTT only 1.2-fold. The dose of heparin required to p...

Thrombosis and Haemostasis

SummaryRo 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following IV administratio... more SummaryRo 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following IV administration to rhesus monkeys, the (mean ± SD.) clear ance, volume of distribution and terminal half-life of Ro 44-3888 were 4.4 ± 1.8 ml/min/kg, 0.8 ± 0.4 l/kg and 2.5 ± 0.8 h respectively. Oral administration of Ro 48-3657 (1 mg/kg), a doubly protected prodrug form, produced peak concentrations of Ro 44-3888 (152 ± 51 ng/ml), 4.2 ± 2.2 h after dosing. Terminal half-life and estimated bioavailabil ity were 5.1 ± 1.6 h and 33 ± 6% respectively. No effect on blood pressure, heart rate or platelet counts were seen. Adenosine diphosohate (ADP) induced platelet aggregation (PA) and cutaneous bleeding times (CBT) were determined prior to and after the last of 8 daily oral administrations of Ro 48-3657 (0.25 or 0.5 mg/kg) to eight rhesus monkeys. Peak and trough plasma concentrations were proportional to dose and steady state was achieved after the second administration. Inhibition of PA and prolongatio...

Thrombosis and haemostasis, 1996

Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acut... more Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH...

New Therapeutic Agents In Thrombosis And Thrombolysis, Revised And Expanded, 2002

Thrombosis and haemostasis, 2001

The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which... more The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which are important for macromolecular substrate activation by the tissue factor:factor VIIa complex. Replacement of these two residues with alanine in a soluble version of human tissue factor resulted in a mutant, hTFAA, which can bind factor VIIa but forms an enzymatically inactive complex. We found that hTFAA inhibits the activity of guinea pig factor VIIa, allowing us to evaluate hTFAA's effects on thrombosis and hemostasis in a guinea pig model of recurrent arterial thrombosis. In addition to heparin, the effects of hTFAA were compared to active site inhibited factor IXa (F.IXai) and factor Xa (F.Xai). We found that hTFAA, F.IXai and F.Xai were potent antithrombotics and may possess a decreased risk of hemorrhage when compared to unfractionated heparin. When administered at a dose that inhibited thrombosis by about 90%, hTFAA neither affected cuticle bleeding nor the activated partia...

Blood, 1997

One approach to developing safer and more efficacious agents for the treatment of thrombotic dise... more One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the acti...

Thrombosis and haemostasis, Jan 2, 1993

In the accompanying paper, we reported that the properties of decreased plasma clearance rate, in... more In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on pla...

Thrombosis and haemostasis, Jan 2, 1993

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycos... more Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clea...

The American journal of clinical nutrition, 1983

The purpose of this study was to evaluate the effect of age on the rate of recovery from iron def... more The purpose of this study was to evaluate the effect of age on the rate of recovery from iron deficiency and red cell loss anemia in the rat. Iron-deficient diets and exchange transfusions with plasma were used to decrease the hematocrit by about 40% below the control value. The results showed progressively slower reversal of the anemia of red cell loss with increasing age. Hematocrit reached control values after 5, 10, and 14 days, at 50, 100, and 360 days of age, respectively. In contrast, iron-deficiency anemia was reversed at similar rates after intravenous treatment with iron dextran; control hematocrit values were reached after 4 and 5 days in the 50-and 100-day-old rats, respectively. The results indicate that the rate of reversal of the anemia of red cell loss is more age-dependent than the rate of reversal of iron-deficiency anemia in the male rat.

The American journal of physiology, 1984

Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl... more Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl in controls) and decreased oxidative capacities of skeletal muscle (as much as 90% below control values). Whole-animal maximal O2 consumption (VO2max), measured in a brief treadmill run of progressively increasing work load, was approximately 50% lower for iron-deficient rats than for controls, and maximal endurance capacity (time to exhaustion in a separate treadmill run at a constant, sub-Vo2max work load) was 90% lower for iron-deficient rats than for controls. Exchange transfusion, with packed erythrocytes or plasma, was used to adjust Hb to an intermediate concentration of approximately 9.5 g/dl in both iron-deficient and and control rats. This procedure corrected the Vo2max of iron-deficient rats to within 15% of control values, whereas endurance capacity showed no improvement. Our experimental dissociation of Vo2max and endurance capacity provides further evidence that Vo2max is ...

The Journal of nutrition, 1984

This study was designed to determine the ACTH-corticosterone response to two types of stress in r... more This study was designed to determine the ACTH-corticosterone response to two types of stress in relation to the daily rhythm of food and water intake in the iron-deficient rat. Rats were fed diets containing 2, 10 or 50 mg iron/kg diet between weaning at 21 days and the stress experiments at 38 or 42 days of age. The two iron-deficient diets (2 and 10 mg iron/kg) resulted in mean hemoglobin concentrations of about 6.0 and 8.5 g/dl, respectively, in contrast to about 12.5 g/dl in the control group receiving 50 mg iron/kg diet. Food and water consumption followed the normal nocturnal pattern, irrespective of iron intake. ACTH and corticosterone showed the normal baseline peaks at the 2000 hour lights-out point in all groups. Responses to handling and placement into another cage were similar in most respects but suggested an inappropriately low corticosterone response despite high ACTH values only at the 2000 hour point. However, there was no evidence of similar differences in response...

Ciba Foundation symposium, 1970

The Journal of biological chemistry, Jan 25, 1971

Proceedings of the National Academy of Sciences, 1994

Current treatment with tissue painogen activator (tPA) requires an Intravenous infusion (1.5-3 h)... more Current treatment with tissue painogen activator (tPA) requires an Intravenous infusion (1.5-3 h) became the clearance of tPA from the circulation is rapid (t1/2 6 min). We have developed a tPA variant, T103N,N117Q,

Medicine & Science in Sports & Exercise, 1985

Thrombosis and Haemostasis

SummaryClinical experience suggests that thrombolytic-induced bleeding is associated with systemi... more SummaryClinical experience suggests that thrombolytic-induced bleeding is associated with systemic activation of the thrombolytic system. Using fibrin specific variants of tissue-type plasminogen activator (t-PA) and making use of the apparent fibrin specificity of streptokinase (SK) in the rabbit we tested the hypothesis that minimizing systemic plasmin production and fibrinogenolysis will decrease hemorrhages in models of peripheral bleeding and embolic stroke. t-PA consumed 51% of the available fibrinogen; caused cerebral bleeds and increased peripheral bleeding time. Fibrin-specific variants of t-PA depleted less than 20% of the fibrinogen and did not cause peripheral or cerebral bleeding. However, an equipotent dose of SK converted only 12% of the available fibrinogen but increased bleeding time and caused hemorrhagic conversion in 75% of embolic stroke model animals treated. The data suggest that bleeding associated with tissue-type plasminogen activators is linked to systemic...

Thrombosis and Haemostasis

Summary10C12, a human antibody F(ab’)2, which specifically binds to the Gla domain of factor IX, ... more Summary10C12, a human antibody F(ab’)2, which specifically binds to the Gla domain of factor IX, interfered with all known coagulation processes that involve factor IX/IXa. These include the function of the intrinsic Xase complex and the activation of zymogen factor IX by factor XIa and by the tissue factor:factor VIIa complex. Furthermore, 10C12 potently inhibited activated partial thromboplastin clotting times (APTT) in plasma of guinea pig and rat, thus enabling in-vivo evaluation. In guinea pigs, a bolus administration of 10C12 (10 μg/kg) prevented cyclic flow variations in damaged carotid arteries without affecting coagulation or bleeding parameters. At a 100-fold higher dose, 10C12 had no effect on normal hemostasis as assessed by the cuticle bleeding time. At this dose, 10C12 was also efficacious in a rat arterial thrombosis model, substantially reducing clot weight and duration of vessel occlusion while prolonging ex-vivo APTT only 1.2-fold. The dose of heparin required to p...

Thrombosis and Haemostasis

SummaryRo 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following IV administratio... more SummaryRo 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following IV administration to rhesus monkeys, the (mean ± SD.) clear ance, volume of distribution and terminal half-life of Ro 44-3888 were 4.4 ± 1.8 ml/min/kg, 0.8 ± 0.4 l/kg and 2.5 ± 0.8 h respectively. Oral administration of Ro 48-3657 (1 mg/kg), a doubly protected prodrug form, produced peak concentrations of Ro 44-3888 (152 ± 51 ng/ml), 4.2 ± 2.2 h after dosing. Terminal half-life and estimated bioavailabil ity were 5.1 ± 1.6 h and 33 ± 6% respectively. No effect on blood pressure, heart rate or platelet counts were seen. Adenosine diphosohate (ADP) induced platelet aggregation (PA) and cutaneous bleeding times (CBT) were determined prior to and after the last of 8 daily oral administrations of Ro 48-3657 (0.25 or 0.5 mg/kg) to eight rhesus monkeys. Peak and trough plasma concentrations were proportional to dose and steady state was achieved after the second administration. Inhibition of PA and prolongatio...

Thrombosis and haemostasis, 1996

Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acut... more Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH...

New Therapeutic Agents In Thrombosis And Thrombolysis, Revised And Expanded, 2002

Thrombosis and haemostasis, 2001

The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which... more The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which are important for macromolecular substrate activation by the tissue factor:factor VIIa complex. Replacement of these two residues with alanine in a soluble version of human tissue factor resulted in a mutant, hTFAA, which can bind factor VIIa but forms an enzymatically inactive complex. We found that hTFAA inhibits the activity of guinea pig factor VIIa, allowing us to evaluate hTFAA's effects on thrombosis and hemostasis in a guinea pig model of recurrent arterial thrombosis. In addition to heparin, the effects of hTFAA were compared to active site inhibited factor IXa (F.IXai) and factor Xa (F.Xai). We found that hTFAA, F.IXai and F.Xai were potent antithrombotics and may possess a decreased risk of hemorrhage when compared to unfractionated heparin. When administered at a dose that inhibited thrombosis by about 90%, hTFAA neither affected cuticle bleeding nor the activated partia...

Blood, 1997

One approach to developing safer and more efficacious agents for the treatment of thrombotic dise... more One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the acti...

Thrombosis and haemostasis, Jan 2, 1993

In the accompanying paper, we reported that the properties of decreased plasma clearance rate, in... more In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on pla...

Thrombosis and haemostasis, Jan 2, 1993

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycos... more Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clea...

The American journal of clinical nutrition, 1983

The purpose of this study was to evaluate the effect of age on the rate of recovery from iron def... more The purpose of this study was to evaluate the effect of age on the rate of recovery from iron deficiency and red cell loss anemia in the rat. Iron-deficient diets and exchange transfusions with plasma were used to decrease the hematocrit by about 40% below the control value. The results showed progressively slower reversal of the anemia of red cell loss with increasing age. Hematocrit reached control values after 5, 10, and 14 days, at 50, 100, and 360 days of age, respectively. In contrast, iron-deficiency anemia was reversed at similar rates after intravenous treatment with iron dextran; control hematocrit values were reached after 4 and 5 days in the 50-and 100-day-old rats, respectively. The results indicate that the rate of reversal of the anemia of red cell loss is more age-dependent than the rate of reversal of iron-deficiency anemia in the male rat.

The American journal of physiology, 1984

Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl... more Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl in controls) and decreased oxidative capacities of skeletal muscle (as much as 90% below control values). Whole-animal maximal O2 consumption (VO2max), measured in a brief treadmill run of progressively increasing work load, was approximately 50% lower for iron-deficient rats than for controls, and maximal endurance capacity (time to exhaustion in a separate treadmill run at a constant, sub-Vo2max work load) was 90% lower for iron-deficient rats than for controls. Exchange transfusion, with packed erythrocytes or plasma, was used to adjust Hb to an intermediate concentration of approximately 9.5 g/dl in both iron-deficient and and control rats. This procedure corrected the Vo2max of iron-deficient rats to within 15% of control values, whereas endurance capacity showed no improvement. Our experimental dissociation of Vo2max and endurance capacity provides further evidence that Vo2max is ...

The Journal of nutrition, 1984

This study was designed to determine the ACTH-corticosterone response to two types of stress in r... more This study was designed to determine the ACTH-corticosterone response to two types of stress in relation to the daily rhythm of food and water intake in the iron-deficient rat. Rats were fed diets containing 2, 10 or 50 mg iron/kg diet between weaning at 21 days and the stress experiments at 38 or 42 days of age. The two iron-deficient diets (2 and 10 mg iron/kg) resulted in mean hemoglobin concentrations of about 6.0 and 8.5 g/dl, respectively, in contrast to about 12.5 g/dl in the control group receiving 50 mg iron/kg diet. Food and water consumption followed the normal nocturnal pattern, irrespective of iron intake. ACTH and corticosterone showed the normal baseline peaks at the 2000 hour lights-out point in all groups. Responses to handling and placement into another cage were similar in most respects but suggested an inappropriately low corticosterone response despite high ACTH values only at the 2000 hour point. However, there was no evidence of similar differences in response...

Ciba Foundation symposium, 1970

The Journal of biological chemistry, Jan 25, 1971

Proceedings of the National Academy of Sciences, 1994

Current treatment with tissue painogen activator (tPA) requires an Intravenous infusion (1.5-3 h)... more Current treatment with tissue painogen activator (tPA) requires an Intravenous infusion (1.5-3 h) became the clearance of tPA from the circulation is rapid (t1/2 6 min). We have developed a tPA variant, T103N,N117Q,

Medicine & Science in Sports & Exercise, 1985