Determining the optimal storage time and temperature for performing platelet function assays and global hemostasis assays.

Platelet function assays and global haemostasis assays are essential in diagnosing bleeding tendencies, with light transmission aggregometry (LTA) as golden standard. The Multiple Electrode Aggregation (Multiplate), platelet function assay (PFA) and rotational thromboelastometry (ROTEM) are mostly used as whole-blood screening tests. Currently, patients have to travel to specialized laboratories to undergo these tests, since specific expertise is required. Pre-analytical variables, like storage time and temperature during transport, are still considered to be the most vulnerable part of the process and may lead to discrepancies in the test results. We aim to give a first impression on the stability of blood samples from healthy volunteers during storage and investigate the effect of storage time (1, 3, 6 and 24 hours) and temperature (4°C, room temperature and 37°C) on the Multiplate, PFA, ROTEM and LTA test results. Our data indicated that, for the PFA, whole blood can be stored for 3 hours at room temperature. Whole blood used for the Multiplate and ROTEM can be stored for 6 hours of storage. For LTA, PRP and whole blood were stable up to 3 hours at 4°C or room temperature and 6 hours at room temperature, respectively.

Antithrombotic therapy in high-risk patients after percutaneous coronary intervention; study design, cohort profile and incidence of adverse events.

BACKGROUND: Patients with multiple clinical risk factors are a complex group in whom both bleeding and recurrent ischaemic events often occur during treatment with dual/triple antithrombotic therapy after percutaneous coronary intervention. Decisions on optimal antithrombotic treatment in these patients are challenging and not supported by clear guideline recommendations. A prospective observational cohort study was set up to evaluate patient-related factors, platelet reactivity, genetics, and a broad spectrum of biomarkers in predicting adverse events in these high-risk patients. Aim of the current paper is to present the study design, with a detailed description of the cohort as a whole, and evaluation of bleeding and ischaemic outcomes during follow-up, thereby facilitating future research questions focusing on specific data provided by the cohort. METHODS: We included patients with ≥ 3 predefined risk factors who were treated with dual/triple antithrombotic therapy following PCI. We performed a wide range of haemostatic tests and collected all ischaemic and bleeding events during 6-12 months follow-up. RESULTS: We included 524 high-risk patients who underwent PCI within the previous 1-2 months. All patients used a P2Y12 inhibitor (clopidogrel n = 388, prasugrel n = 61, ticagrelor n = 75) in combination with aspirin (n = 397) and/or anticoagulants (n = 160). Bleeding events were reported by 254 patients (48.5%), necessitating intervention or hospital admission in 92 patients (17.5%). Major adverse cardiovascular events (myocardial infarction, stroke, death) occurred in 69 patients (13.2%). CONCLUSION: The high risk for both bleeding and ischaemic events in this cohort of patients with multiple clinical risk factors illustrates the challenges that the cardiologist faces to make a balanced decision on the optimal treatment strategy. This cohort will serve to answer several future research questions about the optimal management of these patients on dual/triple antithrombotic therapy, and the possible value of a wide range of laboratory tests to guide these decisions.

Thrombin Generation as a Method to Identify the Risk of Bleeding in High Clinical-Risk Patients Using Dual Antiplatelet Therapy.

Background: Patients using dual antiplatelet therapy after percutaneous coronary intervention are at risk for bleeding. It is currently unknown whether thrombin generation can be used to identify patients receiving dual antiplatelet therapy with increased bleeding risk. Objectives: To investigate whether thrombin generation measurement in plasma provides additional insight into the assessment of bleeding risk for high clinical-risk patients using dual antiplatelet therapy. Methods: Coagulation factors and thrombin generation in platelet-poor plasma were measured in 93 high clinical-risk frail patients using dual antiplatelet therapy after percutaneous coronary intervention. During 12-month follow-up, clinically relevant bleedings were reported. Thrombin generation at 1 and 6 months after percutaneous coronary intervention was compared between patients with and without bleeding events. Results: One month after percutaneous coronary intervention, the parameters of thrombin generation, endogenous thrombin potential, peak height, and velocity index were significantly lower in patients with bleeding in the following months compared to patients without bleeding. At 6 months follow-up, endogenous thrombin potential, peak height, and velocity index were still (significantly) decreased in the bleeding group as compared to non-bleeders. Thrombin generation in the patients' plasma was strongly dependent on factor II, V, and VIII activity and fibrinogen. Conclusion: High clinical-risk patients using dual antiplatelet therapy with clinically relevant bleeding during follow-up show reduced and delayed thrombin generation in platelet-poor plasma, possibly due to variation in coagulation factors. Thus, impaired thrombin-generating potential may be a "second hit" on top of dual antiplatelet therapy, increasing the bleeding risk in high clinical-risk patients. Thrombin generation has the potential to improve the identification of patients using dual antiplatelet therapy at increased risk of bleeding.

Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline.

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

Determining the maximal storage time of centrifuged citrated samples for performing add-on routine coagulation tests.

BACKGROUND: Several routine coagulation tests have been developed to give insight in the coagulation pathway. The laboratory diagnostical process consists of 3 phases, the pre-analytical, analytical and post-analytical phase; however, the pre-analytical phase is most sensitive to errors. The amount of time blood is stored, can affect the measurements of these coagulation tests and result in an incorrect conclusion. Therefore, we performed experiments to determine the maximal storage time, centrifuged blood samples can be reliably measured. METHODS: Citrated whole blood from hospital patients, who were tested for routine coagulation, was collected in 2.7 mL citrate tubes. These whole blood samples were centrifuged and the plasma was stored on top of the cell pellet at room temperature. After 2 h, 4 h, 6 h, 12 h and 24 h, the prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time were measured using Sysmex CS2100 coagulation analysers. RESULTS AND CONCLUSION: Analytical evaluation of routine coagulation tests resulted in various significant differences and large variations between the various time intervals. Our results indicated that the PT and INR can be measured up till 24 h of storage. Centrifuged blood for measuring the fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time can be stored up to 4 h, while 2 h of storage might already be too long for obtaining reliable aPTT measurements.

The diagnostic accuracy of bleeding assessment tools for the identification of patients with mild bleeding disorders: A systematic review.

INTRODUCTION: The bleeding assessment tool (BAT) has been developed to standardize and interpret bleeding history for mild bleeding disorders. However, a critical appraisal addressing the quality and results of validation studies is lacking. AIM: We performed a systematic review of diagnostic studies assessing the performance of the BAT in patients referred for evaluation of bleeding symptoms. METHODS: The electronic database PubMed was searched from inception through July 27, 2017. Eligible publications were original studies that assessed and validated the diagnostic accuracy of bleeding questionnaires for identification of adults with mild bleeding disorders. For each study, sensitivity, specificity and diagnostic odds ratio (DOR) were calculated. Quality was assessed using the Quality Assessment of Diagnostic studies-2 tool. To assess the influence of specific study characteristics on DOR, univariate meta-regression analyses were performed. RESULTS: Nine studies were included. Five studies investigating the ISTH-BAT or other bleeding questionnaires had a moderate to low DOR. Four studies investigating Vicenza-based BATs had a high DOR, with high specificity (>90%) and sensitivity of 59%-85%. Study characteristics such as case-control design, retrospective data collection and differences in reference standard were associated with optimistic estimates of diagnostic performance. Three of four studies with a high DOR had these study characteristics. Studies with good methodological quality mainly had a low DOR. CONCLUSION: The main advantage of the BAT is that it offers a complete and structured interview. However, the BAT is of limited diagnostic value to the workup of patients referred for bleeding evaluation in clinical practice.

Direct oral anticoagulants: When to consider laboratory testing?

INTRODUCTION: Direct oral anticoagulants (DOACs) are increasingly prescribed for prevention of thromboembolic stroke, as well as for prevention and treatment of venous thromboembolism. Dose adjustment based on laboratory testing is not required; however, there are several potential situations that deserve insight into a DOAC plasma activity level. METHODS: Based on a series of real-life case descriptions, we discuss indications for dedicated DOAC testing, as well as the interpretation and consequences. RESULTS: Testing of DOACs in selected patients may help to better interpret acute situations such as bleeding or thrombosis while on anticoagulation, but also suspected drug failure, drug accumulation, or lack of adherence. CONCLUSION: The 24/7 availability of target-specific tests with adequate calibration is recommended to support the clinician in the interpretation and where needed adjustment of the management of patients on DOACs. The relevance of laboratory-guided DOAC management, particularly in the elderly, merits further study.

Atherothrombosis and Thromboembolism: Position Paper from the Second Maastricht Consensus Conference on Thrombosis.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

[Haemolysis: role of the direct antiglobulin test and eluate]. / Hemolyse: rol van directe antiglobulinetest en eluaat.

Haemolytic anaemia is the result of an abnormal breakdown of red blood cells. The direct antiglobulin test (DAT), also known as the direct Coombs test, can be used to determine the cause of the haemolysis. The DAT distinguishes between immune and non-immune causes of haemolysis. However, the DAT should not be used in screening for haemolysis. When the DAT is performed without an indication for in vivo haemolysis, there is a high risk of false-positive results. To increase the specificity of the DAT, the eluate can be tested to determine the specificity of the autoantibodies. In this article we present two cases of haemolytic anaemia in which the DAT gives further indication of the cause of haemolysis.

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy.

INTRODUCTION: Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. AIM: In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. MATERIALS AND METHODS: In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbß3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. RESULTS: Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbß3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbß3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. CONCLUSIONS: Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

BACKGROUND AND OBJECTIVES: In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied. MATERIALS AND METHODS: 10 Omniplasma and 10 quarantine plasma units were used to study different procoagulation, anticoagulation and fibrinolytic parameters. Analysis took place at different time-points during plasma storage at 2-6°C. RESULTS: At baseline, significant reduced levels of factor V, free protein S, α2-antiplasmin and tPA-induced ROTEM lysis time were observed in Omniplasma as compared to quarantine plasma. Moreover, thrombin generation, IXa-AT complex levels and factor XIa were significantly increased in Omniplasma. The majority of the parameters studied remained stable in Omniplasma 48 h after thawing, with the exception of factor VIII (decrease) and IXa-AT (increase). CONCLUSION: Our results suggest an increased coagulation potential, presumingly as a result of contact activation during the production process and also, an increased fibrinolytic potential in Omniplasma. The stability of Omniplasma, based upon the different parameters studied, is comparable to Q-plasma. A maximum post-thawing time of 48 hfor Omniplasma can be suggested.

Peri-partum reference ranges for ROTEM(R) thromboelastometry.

BACKGROUND: Post-partum haemorrhage (PPH) causes rapidly developing deficiencies in clotting factors and contributes to substantial maternal morbidity and mortality. Rotational thromboelastometry (ROTEM(®)) is increasingly used as a point of care coagulation monitoring device in patients with massive haemorrhage; however, there are limited data on reference ranges in the peri-partum period. These are required due to the haemostatic changes in pregnancy. METHODS: In a Dutch multi-centre trial, 161 subjects were included; blood samples were obtained during labour (T1) and within 1 h of delivery (T2). Reference ranges of ROTEM(®) INTEM, EXTEM, FIBTEM, and APTEM were set and correlation with laboratory results was investigated using the guidelines of the International Federation of Clinical Chemistry. RESULTS: Reference ranges were obtained for clotting time (CT), clot formation time (CFT), α-angle, clot firmness at 10 and 20 min (A10, A20), maximum clot firmness (MCF), and maximum lysis (ML). These were comparable from centre to centre, and between T1 and T2. Reference ranges T1: EXTEM: CT 31-63 s, CFT 41-120 s, and MCF 42-78 mm. INTEM: CT 109-225 s, CFT 40-103, and MCF 63-78 mm. FIBTEM: CT 31-79 s and MCF 13-45 mm. APTEM: CT 33-62 s, CFT 42-118, and MCF 61-79 mm. CONCLUSIONS: Reference values for ROTEM(®) parameters are reported. The previously published correlation between FIBTEM parameters and plasma fibrinogen levels by the Clauss method is confirmed. Further research is needed to define threshold values for haemostatic therapy in the course of PPH. Clinical trial registration NTR 2515 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2515).

Triple red blood cell alloantibody formation after bone-allograft transplantation.

In this case report, we provide evidence for the possibility of red blood cell alloimmunization after bone-allograft transplantation. Here, we present a 13-year-old boy who received a bone allograft due to impending hip-luxation. Five months later he was shown to have developed three different alloantibodies: anti-D, anti-C and anti-E, which were induced by the bone allograft. Red blood cell alloimmunization is a possible adverse event when a patient is exposed to allogenic red blood cells. These antibodies may cause transfusion reactions when incompatible blood is administered. More importantly, these antibodies may cause severe, or even fatal, hemolytic disease of the fetus or newborn, stretching the importance of preventing antibody formation, especially in young women. This case demonstrates the importance of selecting rhesus phenotype compatible bone allografts.

Platelet concentrate transport in pneumatic tube systems--does it work?

Blood sample transport via pneumatic tube systems (PTS) reduces the turnaround time of laboratories, but it might influence analysis results. Its effect on platelet concentrates (PCs) is not known. Platelet function was investigated after single and multiple PTS transport in comparison with storage and irradiation. Optical and impedance aggregation, CD-62, and microparticles changed as a result of storage, but not due to transport. Irradiation lowered platelet function independently. Multiple transport impaired thrombin receptor-activating peptide-induced aggregation. This investigation demonstrates the feasibility of PTS transport. As platelet function depends on storage, it may be more important to transfuse fresh PCs.

Acute effects of hemodialysis on salivary flow rate and composition.

AIMS: To evaluate acute effects of hemodialysis (HD) on the salivary flow rate, pH and biochemical composition before, during and after completion of a dialysis session. MATERIAL AND METHODS: Unstimulated whole saliva (UWS) and chewing-stimulated whole saliva (CH-SWS) were collected in 94 HD patients. Salivary flow rate, pH, concentrations of total protein, albumin, cystatin C, secretory immunoglobulin A (S-IgA) and of sodium, potassium and urea were measured. RESULTS: HD had an acute stimulating effect on the salivary flow rate (UWSbefore = 0.30+/-0.22 ml/min, UWSduring = 0.39+/-0.25 ml/min, p < 0.005). The mean pH of UWS showed a small but significant increase during HD mainly due to an increased watery secretion from the salivary glands. The salivary biochemical constituents changed markedly, but no significant difference in output was found. The electrolyte concentration did not change significantly during dialysis. The level of urea in CH-SWS declined to 40% (Ureabefore = 25.+/-6.4 mmol/l, Ureaduring = 15.3+/-4.5 mmol/1). CONCLUSIONS: This study shows that HD has significant acute effects on both salivary secretion rate and protein concentrations in saliva. We conclude that the observed changes in salivary concentrations and proteins are mainly due to an increased watery secretion from the salivary glands.

Salivary cystatin activity and cystatin C in natural and experimental gingivitis in smokers and non-smokers.

BACKGROUND: Recent studies show that subjects with natural gingivitis or periodontitis have elevated levels of salivary cystatins compared to periodontally healthy individuals. Increased glandular output of cystatins in inflammatory conditions suggests an active, most likely protective, rôle for these proteins in inflammatory processes. Furthermore, it has been shown that the development of gingival inflammation is suppressed in smokers during experimental gingivitis. AIMS: The purpose of the present study was to investigate whether (i) the levels of salivary cystatins in natural gingivitis are related to smoking status, and (ii) to study whether experimentally induced gingivitis is associated with changes in salivary cystatin levels, in both smokers and non-smokers. MATERIAL AND METHODS: Whole saliva samples were taken in relation to natural gingivitis, gingival health and 14-day experimental gingivitis in 25 non-dental students (14 non-smokers and 11 smokers). The salivary flowrate was determined. Samples were analyzed for levels of protein, cystatin and cystatin-C. RESULTS: Salivary flow and protein concentrations in cleared human whole saliva samples of non-smokers and smokers were not different from each other at any timepoint during the trial. With regard to cystatins, the results showed that in the state of natural gingivitis cystatin activity is lower in smokers as compared to non-smokers. In smokers, the resolution of natural gingivitis to the state of gingival health did not result in a change of cystatin activity and levels of cystatin C. At the end of the 14-day experimental gingivitis period, smokers showed a decrease in cystatin activity and cystatin C as well as lower outputs of cystatin activity and cystatin C. CONCLUSION: Smoking is associated with lower cystatin activity and output of cystatin C during gingival inflammation.

Cystatin A in gingival crevicular fluid of periodontal patients.

Cystatins are physiological inhibitors of cysteine proteinases which are widely distributed in human tissues and fluids. In the present study we analysed both the cystatin activity and the different cystatin isoforms in gingival crevicular fluid and saliva samples of nine periodontitis patients. All crevicular fluid samples, which were collected with filter paper points, showed cystatin activity ranging from 7-67 units/mg protein. The mean cystatin activity (24 units/mg protein) was significantly lower (p < 0.05) than that of the saliva samples (mean 93 units/mg protein). The cystatin isoforms in the crevicular fluid were further characterized by immunoblotting with specific antibodies against cystatin C, S, SN and A. While they were clearly present in saliva, cystatin C, cystatin S and cystatin SN could not be detected in any of the crevicular fluid samples. Remarkably, cystatin A was found in all the crevicular fluids as well as in the saliva samples. It is concluded that the cystatin activity found in crevicular fluid is caused, at least partially, by cystatin A. Furthermore, the gingival crevicular fluid is not a major contributor of cystatin C, S and SN activity in saliva.

Inhibition of the growth and cysteine proteinase activity of Porphyromonas gingivalis by human salivary cystatin S and chicken cystatin.

The mutual effects of P. gingivalis and several cystatin species has been investigated. After incubation with P. gingivalis culture supernatant, cystatin S, cystatin C and chicken cystatin were truncated from a 14 kDa protein into a polypeptide of approximately 13 kDa. Amino acid sequence analysis of the truncated cystatin S polypeptide revealed that cystatin S was cleaved after Arg-8. All three types of truncated cystatins fully retained their inhibitory activity toward papain. Cystatin S and chicken cystatin partially inhibited proteolytic activity in the culture supernatant of P. gingivalis. Furthermore, cystatin S and chicken cystatin inhibited the growth of P. gingivalis in culture to 50% at approximately 1 microM.