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J Henkin

Publications and source records attributed to J Henkin.

At least 55 records · Page 3Linked to original sources
PubMed1992

A chromogenic enzymatic assay capable of detecting prourokinase-like material in plasma.

We report a functional assay capable of quantifying prourokinase (ProUK)- like material in plasma where urokinase (UK) is also present. The assay involves inactivation of urokinase with a specific, active site directed irreversible inhibitor, dansyl-glutamyl glycyl arginine chloromethylketone (dansyl- GGACK). Excess inhibitor is subsequently quenched with dithiothreitol (DTT). The ProUK-like material in plasma is then converted to active urokinase with thermolysin, a proteolytic enzyme of bacterial origin. Alpha 2-macroglobulin in plasma inhibits thermolysin; however alpha 2-macroglobulin is inactivated with methylamine. The assay can detect as little as 20 ng of ProUK and is linear from 20 to 120 ng. The assay was applied to quantify the amount of ProUK-like material in plasma obtained from dog at various times after i.v. administration of 100,000 or 75,000 U/kg, of pro-urokinase.

Amino Acid Chloromethyl Ketones↗
PubMed1992

New developments in thrombolytic therapy.

Pharmacologic lysis of occlusive, ischemia-producing thrombi has become widely accepted during the past decade. New developments in this field have centered around increasing the efficacy of the known plasminogen activators while employing methods to minimize the risk of hemorrhage and decrease the incidence of rethrombosis. Such methods have included the use of thrombus-directed antibodies linked to plasminogen activators, increased plasminogen (substrate) concentration at the thrombus site, anticoagulant and antiplatelet therapy to prevent thrombus propagation and reformation following lysis, and combination plasminogen activator therapy designed to increase efficacy and safety. These new strategies have been extensively tested in vitro and in a variety of animal models. As we have indicated, extrapolation of such results to human patients cannot be done with confidence. However, the strategies are based on sound rationale and the reported findings should serve as the basis for controlled human trials.

Humans↗
PubMed1992

Approach to the patient with venous thromboembolism. Treatment with thrombolytic agents.

This article reviews the different thrombolytic agents currently available and the different mechanisms by which they activate the body's fibrinolytic system. The discussion is confined to the approach to the patient with venous thromboembolism using the different thrombolytic agents. Data are presented supporting the use of thrombolytic therapy and its long-term benefits, especially in patients with pulmonary embolism. A substantial portion of this article is devoted to practical considerations involved in the administration of thrombolytic therapy.

Amino Acid Sequence↗
PubMed1991

igh sialic acid content slows prourokinase turnover in rabbits.

The clearance of natural and recombinant prourokinase (proUK) from the blood of rabbits was studied by means of a double-isotope method which allowed the differential removal of two distinct proUK species to be monitored when simultaneously administered to an individual animal. In initial experiments, proUK expressed in different cell lines contained between 0 and 2.5 molecules of sialic acid per molecule of protein. A slight trend toward slower clearance of proUK with higher sialic acid content was observed but rate differences were not statistically significant. Recombinant proUK produced in CHO cells grown in flow reactors, contained unusually high levels of sialic acid in excess of 3 moles/mole protein. Controlled exposure to immobilized neuraminidase was used to remove sialic acid from this protein in defined amounts. The clearance of the parent material was biphasic with average alpha and beta half-lives of 1.7 min and 16.7 min respectively. The AUC of the parent material was only slightly lowered upon removal of 30% of the original sialic acid. Species with 60% or 90% removal of sialate were much more rapidly cleared from the circulation respectively yielding AUCs equal to 56% and 41% of that observed with the parent material. Thus proUK containing 2.5-3.5 sialic acid molecules per molecule of protein turned over significantly more slowly in rabbits than did less sialylated proUK. The clearance rate was relatively insensitive to sialic acid content between 0 and 1.5 sialic acid residues per proUK molecule.

Animals↗
PubMed1991

Enhancement of the thrombolytic efficacy of prourokinase by lys-plasminogen in a dog model of arterial thrombosis.

Current findings suggest that the efficacy of thrombolytic therapy may be limited by the availability of active forms of plasminogen at the thrombus site. The purpose of this study was to determine if the systemic administration of 0.5 mg kg-1 glu-plasminogen (glu-plg) or 0.5 mg kg-1 lys-plasminogen (lys-plg) could safely increase the efficacy of a single intravenous bolus injection of 50,000 U kg-1 prourokinase (proUK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by direct visual examination. Forty dogs (mean wt 10.3 +/- 2 kg) were randomly sorted into 4 groups of 10 each. The dogs in each group were given either saline plus saline, saline plus proUK, glu-plg plus proUK, or lys-plg plus proUK 60 minutes after formation of an occlusive arterial thrombus. Ninety minutes after drug administration the dogs receiving saline plus proUK, glu-plg plus proUK, and the lys-plg plus proUK showed greater thrombolysis (41%, 43%, and 66%, respectively) than the control (saline plus saline) group (15%, P less than 0.01). The lys-plg plus proUK treatment caused greater lysis than the saline plus proUK or the glu-plg plus proUK treatment (P less than 0.05). All of the dogs (10/10) receiving lys-plg plus proUK had patent vessels at the end of the 90 minute monitoring period, whereas only 4/10 and 5/10 vessels were patent in the saline plus proUK and glu-plg plus proUK groups, respectively. None of the dogs in the saline plus saline group had patent vessels. No significant changes were observed in the various coagulation parameters tested for any of the 4 treatment groups. The results show that lys-plg can safely increase the thrombolytic efficacy of proUK.

Analysis of Variance↗
PubMed1991

The beneficial effect of lys-plasminogen upon the thrombolytic efficacy of urokinase in a dog model of peripheral arterial thrombosis.

The efficacy of thrombolytic therapy may be limited by local availability of plasminogen near a poorly perfused thrombus. The purpose of this study was to determine if the local (i.e., clot site) administration of 0.5 mg glu-plasminogen (glu-plg) or 0.5 mg lysplasminogen (lys-plg) could safely increase the thrombolytic efficacy of a 30-min intraarterial injection of 3,500 U kg-1 of two-chain urokinase plasminogen activator (UK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by a distally placed flowmeter and by direct visual examination. Forty-two dogs (mean weight 10.1 +/- 1.9 kg) were randomly sorted into six groups of 7 each. The dogs in each group were given either saline plus saline (group 1), saline plus UK (group 2), glu-plg plus saline (group 3), glu-plg plus UK (group 4), lys-plg plus saline (group 5), or lys-plg plus UK (group 6) by selective arterial catheterization 60 min after formation of an occlusive thrombus. Ninety minutes following drug administration, all groups which received UK (groups 2, 4, and 6) showed greater lysis (p less than 0.05) than the groups which received only saline or either glu- or lys-plg plus saline. Group 6, which received lys-plg plus UK, showed significantly greater lysis (34 +/- 4%) than both group 2 (23 +/- 2%), which received saline plus UK, and group 4 (19 +/- 3%), which received glu-plg plus UK (p less than 0.05). All dogs (7/7) in group 6 had reflow at 90 min whereas only 3/7 dogs had reflow in both groups 2 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Involvement of urokinase and its receptor in the invasiveness of human prostatic carcinoma cell lines.

We have investigated the role of urokinase (UK) and its cell-surface receptor in determining the invasiveness of prostate cancer cells. Three human cell lines, DU-145, PC-3 and LNCaP, that differ in androgen-responsiveness and growth characteristics, were tested. Analysis of the conditioned medium by an enzyme-linked immunosorbent assay showed secretion of UK by DU-145 (63 ng/mL/10(6) cells/48 hr) and PC-3 (682 ng/mL/10(6) cells/48 hr), but absence of secretion by LNCaP cells. Western blot analysis and enzyme activity assay of the conditioned medium confirmed these results. Scatchard analysis of radioligand binding with acid pretreated cells showed the presence of a single population of high affinity UK receptors on DU-145 cells (93,000 sites/cell, Kd = 0.9 nM) and PC-3 cells (25,000 sites/cell, Kd = 1.0 nM) but not on LNCaP cells. DU-145 and PC-3 cells were found to be highly invasive in in vitro invasion assays: 4.5 +/- 0.5% and 6.5 +/- 0.5%, respectively, of total tumor cells (approximately 2 x 10(5)) had penetrated reconstituted basement membrane (Matrigel) in a 72 hr incubation in serum-free growth medium. Under similar conditions, less than 0.25% LNCaP cells invaded Matrigel. The data indicate that androgen unresponsive, aggressive prostate tumor cells of high metastatic potential, DU-145 and PC-3, secrete UK and display cell-surface UK receptors, fully charged with the protease. Conversely, relatively indolent LNCaP cells of low metastatic potential do not secrete UK nor do they possess its binding sites. UK receptor antagonists, UK 12-32 and UK 6-135, which compete with labeled UK for binding to prostatic cells but do not inhibit cellular proliferation or UK secretion, markedly reduced DU-145 and PC-3 cell invasion (80-85% inhibition), thereby suggesting an important role of receptor-bound UK in prostate tumor cell invasion.

Basement Membrane↗
PubMed1990

An amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) is mitogenic for osteoblast-like cells.

A peptide mitogen for cultured osteoblast-like cells was purified from serum-free conditioned culture medium of a human prostatic cancer cell line, PC-3. Based on amino acid sequencing and estimated molecular weight, this peptide was identified as an NH2-terminal fragment of urokinase-type plasminogen activator (uPA). Recombinant high molecular weight (HMW) uPA and the NH2-terminal growth factor domain (GFD) of uPA, but not low molecular weight (LMW) uPA (lacking the NH2-terminal region) stimulated [3H] thymidine incorporation and proliferation in osteoblast-like cells, and specific, competitive binding sites for HMW, but not LMW, uPA were demonstrable. These studies demonstrate the production of a mitogenic NH2-terminal fragment of uPA by a human prostatic cancer cell line which may be of importance in the pathogenesis of osteoblastic metastases.

Adenocarcinoma↗
PubMed1990

The protective effect of heparin in a dog model of rethrombosis following pharmacologic thrombolysis.

Rethrombosis is an important clinical problem for patients who have benefitted from pharmacologic thrombolysis. The present study describes a dog model of arterial thrombosis, which includes endothelial denudation, intimal damage, and stenosis, and is suitable for studying the phenomena of both thrombolysis and subsequent rethrombosis. The model was used to determine the effect of tissue-type plasminogen activator (t-PA), high and low dose heparin, and saline upon the incidence of rethrombosis after t-PA-induced thrombolysis. Initial thrombolysis with reflow was achieved with 0.4 mg/kg t-PA, intravenous bolus injection, followed immediately by 0.4 mg/kg t-PA, 30 min infusion, in 40 of 42 dogs (95%) that had an occlusive, 125I-labelled thrombus created in a segment of femoral artery. The 40 dogs in which reperfusion was achieved were randomly sorted into 4 groups of 10 each which then received either saline, t-PA (0.4 mg kg-1 infused over 1 h), low dose heparin (500 U bolus injection then 250 U h-1 for 24 h), or high dose heparin (1,500 U bolus injection then 500 U h-1 for 24 h). Sixty percent (6/10) of the saline treated dogs showed occlusive rethrombosis at 24 h. The incidence of occlusive rethrombosis was 9/10 in the t-PA treated group (p = NS), 3/10 in the low dose heparin treated group (p = NS), and 0/10 in the high dose heparin treated group (p less than 0.01). Two smaller groups consisting of 5 dogs each were treated with either saline or high dose heparin alone (no t-PA). None of the dogs in either group showed thrombolysis with reflow.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA.

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead to an increase in its apparent molecular mass by approximately 40 Kd. The predominant u-PA binding protein isolated from whole cell detergent extracts migrated with a molecular mass of approximately 36 Kd using affinity chromatography. In contrast, when only cell surface proteins were radiolabeled before extraction, the predominant labeled u-PA binding protein isolated migrated with a molecular mass of approximately 46 Kd. Several pieces of evidence suggested that the difference in molecular mass between these two u-PA binding proteins resulted from glycosylation of a single receptor protein. First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36-Kd species was unaffected. Moreover, the apparent molecular mass of purified surface-labeled receptor (approximately 46 Kd) was reduced by N-glycanase. These studies suggest that the u-PA receptor on the surface of HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which is similar immunologically to u-PA receptors on other cell types.

Base Sequence↗
PubMed1990

Selective binding of plasmin in the presence of excess plasminogen by certain anionic polystyrene resins.

Contact of plasminogen with sulfonate or sulfonyl-glutamate derivatized polystyrene resins has previously been reported to lead to the formation of an active single-chain form of the plasminogen (Kichenin-Martin et al., Thrombosis Research, 52, 469-478, 1988). Attempts to duplicate this finding revealed instead that these polymers selectively adsorb active plasmin and thus remove it even in the presence of a great excess of plasminogen. Under optimum conditions 4.0 mg/mL plasminogen containing 3% plasmin was freed of one half of this contaminant by exposure to 6-9 mg of dry resin per mL in a batch mode. Neither ordinary polystyrene nor Dowex nor Sephadex cation exchange resins displayed these properties. The separation is based on the more rapid binding of plasmin to the active resins and is thus kinetically controlled.

Anions↗
PubMed1990

Enhanced lytic efficacy of multiple bolus injections of tissue plasminogen activator in dogs.

The purpose of this study was to compare the lytic efficacy of tissue plasminogen activator (tPA) following different dosing regimens. Radiolabelled clots from dog whole blood were inserted into an extracorporeal jugular loop in anesthetized dogs. Clot size (counts/min) was continuously recorded. tPA was administered as a single 1 min bolus of 0.4 mg/kg, as four multiple bolus injections of 0.1 mg/kg each at 30 min intervals, or as an initial bolus (0.04 mg/kg) followed by a 30 min infusion of 0.36 mg/kg (10%/90%, bolus/infusion). Multiple injections of the same total dose of tPA resulted in 76% and 51% greater clot lysis than single bolus injection or bolus/infusion regimen, respectively, measured 120 min after initial dosing. There were no differences in fibrinogen, plasminogen or alpha-2-antiplasmin between the different treatment groups at 120 min.

Animals↗
PubMed1990

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells.

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.

Binding, Competitive↗
PubMed1988

Bolus dose response characteristics of single chain urokinase plasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis.

Tissue plasminogen activator (t-PA) and single chain urokinase-plasminogen activator (scu-PA) are relatively "fibrin-specific" thrombolytic drugs with short plasma half lives of 6-8 minutes. Most treatment regimens with these agents utilize a bolus injection followed by continuous drug infusion, usually combined with anticoagulant therapy. The purpose of this study was to establish the dose-response characteristics for scu-PA and t-PA, when given as a single intravenous bolus injection, in a dog model of arterial thrombosis. Eight groups of 6 dogs each were given one of the following doses of scu-PA (mg/kg): 0.20, 0.50, 1.00, 2.00; or t-PA: 0.05, 0.10, 0.20; or an equivalent amount of saline (control group). All doses were given as a single bolus injection 60 minutes after formation of a totally occlusive femoral artery thrombus. Thrombolysis was measured by monitoring the continuous decrement of 125I activity from a radiolabelled thrombus. Ninety minutes after drug injection, all scu-PA treated dogs showed greater thrombolysis (30%, 45%, 56%, and 67%, respectively) than the control group (15%, p less than 0.01). The 0.10 and 0.20 mg/kg t-PA treated dogs showed greater thrombolysis (35% and 49%, respectively) than the control group (15%, p less than 0.01). Both scu-PA and t-PA caused a partial and dose-dependent decrease in alpha 2-antiplasmin activity but scu-PA caused a greater depletion (72% vs. 18%, respectively, p less than 0.05) at 60 minutes after the highest dose of drug administration. Both drugs showed a longer than expected thrombolytic effect based upon the known half lives. Neither drug caused significant changes in the prothrombin time, activated partial thromboplastin time, thrombin time, hematocrit, platelet count, or fibrin degradation product concentration. Single bolus injections of scu-PA and t-PA produce safe and effective thrombolysis in this dog model of arterial thrombosis.

Animals↗
PubMed1988

Optimizing the bolus/infusion ratio for intravenous administration of urokinase in dogs.

The purpose of this study was to determine the optimal bolus/infusion ratio of urokinase (UK) in a dog model of experimental thrombosis. Radiolabelled clots from dog whole blood were inserted into an extracorporeal jugular loop in anesthetized dogs. Clot size (counts/min) was continuously recorded. UK was administered i.v. as a 1 min bolus (100%/0%), 30 min infusion (0%/100%), or as a bolus/infusion combination at ratios of 75%/25%, 50%/50%, or 25%/75%. The total dose of UK in all groups was 20,000 U/kg. A bolus/infusion ratio of 25%/75% produced twice as much lysis as either bolus or infusion alone. There were no statistical differences between the UK-treated groups in reduction of plasma alpha 2-antiplasmin concentration. We conclude that thrombolytic efficacy was increased by the bolus/infusion administration of UK without a concomitant increase in systemic plasminogen to plasmin conversion.

Animals↗
PubMed1988

Sequence-specific assignments in the 1H NMR spectrum of the human inflammatory protein C5a.

Full sequence-specific assignments for the 1H NMR lines of the backbone protons of the human complement factor C5a are described and documented. The results were obtained by largely following the methodology developed by Wüthrich et al. [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311]. Assignments for the majority of the amino acid side chain protons were obtained by using a comparison of double- and triple-quantum-filtered two-dimensional correlated experiments together with the analysis of relayed coherence transfer spectra. The assignments provide the basis for the determination of the thus far unknown three-dimensional structure of C5a from nuclear Overhauser enhancement distance constraints.

Amino Acid Sequence↗