Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa

1994 ◽  
Vol 267 (2) ◽  
pp. C466-C472 ◽  
Author(s):  
X. Zheng ◽  
M. Geiger ◽  
S. Ecke ◽  
E. Bielek ◽  
P. Donner ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Protein C ◽  
Reproductive Tract ◽  
Human Sperm ◽  
Order Rate Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amidolytic Activity ◽  
Protein C Inhibitor ◽  
Seminal Plasma Proteins ◽  
High Concentrations

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

Amidolytic kinetic assay of protein C by selective spectrophotometry in a centrifugal analyzer.

1988 ◽  
Vol 34 (11) ◽  
pp. 2260-2263 ◽  
Author(s):  
I Kobayashi ◽  
N Amemiya ◽  
T Endo ◽  
J Motegi ◽  
A Kurihara ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Subjects ◽  
Mean Value ◽  
Human Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amidolytic Activity ◽  
Kinetic Assay ◽  
The Difference ◽  
High Concentrations ◽  
Agkistrodon Contortrix Contortrix

Abstract This rapid, simple amidolytic assay of protein C activity in whole plasma involves activation by protein C activator from the venom of Agkistrodon contortrix contortrix (Protac) and use of a Cobas Fara spectrophotometer programmed for kinetic assay. Plasma is incubated with activator venom in the presence or absence of antibody to human protein C in the instrument, chromogenic substrate (S-2366) is added, and the absorbance is measured at 405 nm. The difference between the absorbance of the sample plasma with and without antibody to human protein C correlated well with protein C antigen as assayed by enzyme-linked immunosorbent assay (ELISA) and the Laurell rocket technique in normal subjects, patients being treated with warfarin, and patients with liver cirrhosis or disseminated intravascular coagulation. Our mean value for protein C in normal subjects is 115.9 (SD 16.7)% for amidolytic activity, 103.0 (SD 17.4)% for ELISA, and 97.2 (SD 18.1)% for the rocket technique. The high value for normal subjects presumably includes some nonspecific amidolytic activity activated by the activator venom, as indicated by measurable activity in immuno-depleted protein C-deficient plasma. Within-run and between-run CVs were less than 5% at low, normal, and high concentrations of protein C amidolytic activity.

scholarly journals The Human Cationic Antimicrobial Protein (hCAP-18) Is Expressed in the Epithelium of Human Epididymis, Is Present in Seminal Plasma at High Concentrations, and Is Attached to Spermatozoa

2000 ◽  
Vol 68 (7) ◽  
pp. 4297-4302 ◽  
Author(s):  
Johan Malm ◽  
Ole Sørensen ◽  
Terese Persson ◽  
Margareta Frohm-Nilsson ◽  
Bengt Johansson ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Reproductive System ◽  
Male Reproductive System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antimicrobial Protein ◽  
Healthy Donors ◽  
Standard Deviation Range ◽  
Doublet Band ◽  
High Concentrations

ABSTRACT Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 ± 37.8 μg/ml (mean ± standard deviation; range, 41.8 to 142.8 μg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/106 spermatozoa, corresponding to 6.6 × 106 molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.

scholarly journals Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 722-728 ◽  
Author(s):  
M Geiger ◽  
K Huber ◽  
J Wojta ◽  
L Stingl ◽  
F Espana ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Specific Activity ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Protein C Inhibitor

Abstract Protein C inhibitor (PCI) and plasminogen activator inhibitor 3 (PAI-3; urinary urokinase inhibitor) are immunologically identical. The role of PCI for urokinase (uPA) inhibition in vivo was investigated. We therefore developed an enzyme-linked immunosorbent assay (ELISA) specific for uPA-PCI complexes: Rabbit anti-PCI IgG was immobilized on a microtiter plate and following incubation with uPA-PCI complex- containing samples, bound uPA-PCI complexes were quantified with a horseradish-peroxidase-linked monoclonal antibody (MoAb) to uPA. Using this assay, time, dose, and heparin-dependent complexes were detected when uPA was incubated with normal plasma or purified urinary PCI, whereas no complexes were measurable using PCI-immunodepleted plasma. Plasma samples (containing 20 mmol/L benzamidine to prevent complex formation ex vivo) from patients undergoing systemic urokinase therapy (1 x 10(6) IU/60 min intravenously [IV]) after myocardial infarction were also studied. uPA present in these plasma samples (up to 1,200 ng/mL) had only 43% to 70% of the specific activity of purified 2-chain uPA, suggesting that a major portion of uPA is complexed to inhibitors. In these plasma samples uPA-PCI complexes were present in a concentration corresponding to 21% to 25% of inactive uPA antigen. These data suggest that at high uPA concentrations, such as during uPA therapy, plasma PCI might contribute significantly to uPA inhibition in vivo.

Interaction of Plasma Kallikrein with Protein C Inhibitor in Purified Mixtures and in Plasma

1991 ◽  
Vol 65 (01) ◽  
pp. 046-051 ◽  
Author(s):  
Francisco España ◽  
Amparo Estelles ◽  
John H Griffin ◽  
Justo Aznar
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Room Temperature ◽  
Activated Protein C ◽  
Physiological Role ◽  
Plasma Kallikrein ◽  
Amidolytic Activity ◽  
Normal Plasma ◽  
Protein C Inhibitor ◽  
Subsequent Addition

SummaryThe interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK: PCI) or to APC (APC: PCI) was measured by sandwich ELISA’s using antibodies directed against each protein in the complexes. The formation of KK: PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 °C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK: PCI complexes. PCI activity fell to 35% of the NHp and 1.2 μ/ml of KK: PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 °C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperaturedependent manner. Addition of 2.8 μ/ml KK to plasma at 4 °C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 μ/ml KK: PCI which represents 21% of the KK added, whereas at 37 °C PCI was inactivated to 30% and only 0.30 μg/ml KK: PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 °C. At 37 °C, PCI accounted for aborfi 7% of the inhibition of the KK added. In separate experiments, following addition of 2.5 μg/ml APC to NHR more than 1 μg/ml of APC: PCI complex was formed in 3 h. When NHP was prior incubated with KK, PCI activity decreased to 10% of that of the normal plasma. Subsequent addition of APC to the plasma treated with KK resulted in formation of only 35 μg/ml of APC: PCI complex compared to 1,350 μg/ml when plasma was not previously incubated with KK. These results indicate that PCI could play a physiological role in the inhibition of plasma KK, and that, in turn, plasma KK can either complex to or inactivate plasma PCI. Thus, KK could modulate the PCI inhibition of APC in plasma.

Evidence for the Regulation of Urokinase and Tissue Type Plasminogen Activators by the Serpin, Protein C Inhibitor, in Semen and Blood Plasma

1993 ◽  
Vol 70 (06) ◽  
pp. 0989-0994 ◽  
Author(s):  
Francisco España ◽  
Amparo Estellés ◽  
Pedro J Fernández ◽  
Juan Gilabert ◽  
Jaime Sánchez-Cuenca ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Seminal Plasma ◽  
Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protein C Inhibitor ◽  
Dependent Cell ◽  
Cell Invasiveness

SummarySince the serine protease inhibitor, protein C inhibitor (PCI), is present in seminal plasma at ≈3 μM, complexes of PCI with urokinase (uPA) and tissue type (tPA) plasminogen activator were quantitated using sandwich enzyme-linked immunosorbent assays (ELISA’s). Seminal plasma (N = 10) collected in the absence of extrinsic inhibitors had a mean of 25 ± 5 ng/ml uPA: PCI, 76 ± 23 ng/ml tPA: PCI, and 4 ± 2 ng/ml of tPA complexes with plasminogen activator inhibitor-1 (tPA:PAI-l). 93% of the uPA and 17% of the tPA antigen in seminal plasma was in complex with PCI and, when complexation was inhibited by collecting semen into an 1,10-phenanthrolinium solution, 33% of the uPA and 7% of the tPA was complexed to PCI. Urine (N = 10) contained 4 ± 1 ng/ml uPA:PCI. In purified system, complexation of uPA and tPA to PCI paralleled the inhibition of the enzymes. In vitro studies in blood and seminal plasma showed that heparin stimulated complexation of uPA and tPA with PCI, suggesting that negatively charged glycosaminoglycans in blood vessels and in the reproductive system may regulate PCI reactions with uPA and tPA. These results suggest that PCI is a physiologic regulator of uPA and tPA in male reproductive tissues and raises questions about a potential role of PCI in human fertility and in uPA-dependent cell invasiveness.

scholarly journals C-Terminal Region of Outer Surface Protein C Binds Borreliacidal Antibodies in Sera from Patients with Lyme Disease

2003 ◽  
Vol 10 (4) ◽  
pp. 573-578 ◽  
Author(s):  
Dean A. Jobe ◽  
Steven D. Lovrich ◽  
Ronald F. Schell ◽  
Steven M. Callister
Keyword(s):  
Lyme Disease ◽  
Outer Surface ◽  
Protein C ◽  
Surface Protein ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carboxy Terminus ◽  
Outer Surface Protein ◽  
Serum Titer ◽  
High Concentrations

ABSTRACT Borreliacidal antibodies specific for outer surface protein C (OspC) are induced shortly after infection with Borrelia burgdorferi. In this study, we identified the region of OspC recognized by immunoglobulin M (IgM) and IgG borreliacidal antibodies. Sera from patients with early Lyme disease were screened for borreliacidal activity specific for B. burgdorferi 50772 and OspC antibodies. Seven sera that contained similarly high titers of each response were then chosen randomly and adsorbed with OspC or a truncated OspC (OspC-Dra) containing the 50 amino acids nearest the carboxy terminus. Adsorption with OspC or OspC-Dra completely eliminated the borreliacidal activity in six (86%) of seven sera and significantly decreased the activity in the remaining serum (titer of 10,240 to 1,280). Moreover, OspC antibodies were no longer detected by OspC enzyme-linked immunosorbent assay or in a Western blot that contained native OspC. The findings confirmed that sera from patients with early Lyme disease contain high concentrations of IgM or IgG borreliacidal antibodies that bind a conserved region of OspC.

scholarly journals Ram seminal plasma and its functional proteomic assessment

Reproduction ◽  
2019 ◽  
Vol 157 (6) ◽  
pp. R243-R256 ◽  
Author(s):  
T Leahy ◽  
J P Rickard ◽  
N C Bernecic ◽  
X Druart ◽  
S P de Graaf
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Reproductive Tract ◽  
Reproductive Technologies ◽  
Female Reproductive Tract ◽  
Sperm Function ◽  
Seminal Plasma Proteins ◽  
Ram Sperm

Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa bothin vitroandin vivoby affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and appliedin vitroto prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.

scholarly journals Normal titer of functional and immunoreactive protein-C inhibitor in plasma of patients with congenital combined deficiency of factor V and factor VIII

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1266-1270 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
S Hashimoto ◽  
T Kamiya ◽  
H Saito
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Amidolytic Activity ◽  
Normal Plasma ◽  
Coagulant Activity ◽  
Protein C Inhibitor ◽  
Combined Deficiency ◽  
Good Agreement

Protein-C inhibitor (PCI) is a newly described plasma inhibitor directed against a vitamin-K-dependent serine protease, activated protein-C, which is involved in the inactivation of factor V and factor VIII. Marlar and Griffin have reported that PCI activity is absent in the plasma of patients with congenital combined factor V/VIII deficiency. We have measured the levels of PCI in the plasma of seven unrelated patients with this disorder using both functional and immunologic methods. The rate at which the amidolytic activity of activated protein-C was neutralized in the patients' plasma was essentially identical to that observed in normal plasma. The titer of PCI antigen, as measured by an electroimmunoassay using a monospecific anti-PCI serum, was 5.3 +/- 1.6 micrograms/ml in the patients' plasma and was not significantly different from that of normal plasma (5.3 +/- 2.7 micrograms/ml, n = 30). The levels of factor-V-related antigen, factor V coagulant antigen, and factor VIII coagulant antigen were low in all patient plasma and were in good agreement with their respective coagulant activity. Our results do not appear to support the hypothesis that combined factor V/VIII defect is due to a lack of PCI.

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